WO2005052152A1 - Dna participating in hydroxylation of macrolide compound - Google Patents

Abstract

It is intended to provide a DNA participating in the hydroxylation of a macrolide compound 11107B and a novel method of producing a macrolide compound 11107D. More specifically speaking, a DNA participating in the biological conversion of the macrolide compound 11107B represented by the formula (I) into the 16-hydroxylated macrolide compound 11107D represented by the formula (II) which encodes a protein having a 16-hydroxylase activity or ferredoxin; a method of isolating the same; a protein encoded by the above DNA; a plasmid carrying the DNA; a transformant transformed by the plasmid; and a method of producing a 16-hydroxylated macrolide compound with the use of the transformant.

Description

DNA technology involved in hydroxylation of macrolide compounds

 The present invention relates to a DNA involved in hydroxylation of a macrolide compound, a method for isolating the same, a protein encoded by the DNA, a plasmid carrying the DNA, a transformant transformed with the plasmid, and The present invention also relates to a method for producing a 16-hydroxylated oxalate compound using the transformant. Conventional technology

 Expression (Π)

 11-membered macrolide compound 11107D represented by 11 orchid (ii) is a 12-membered macrolide compound having excellent antitumor activity and is represented by the formula (I)

11107B (I) 7906

Along with a 12-membered ring macrolide compound 11107B, which is found in a culture of Streptomyces sp. Mer-11107 strain (W0

02/060890). Macrolide compound 11107D is equivalent to the hydroxylated compound at position 16 of macrolide compound 11107B, but its productivity is lower than that of macrolide compound 11107B, and it is hoped that an efficient production method will be established. Had been rare. Disclosure of the invention

 An object of the present invention is to find DNA involved in hydroxylation of macrolide compound 11107B, and to provide a novel method for producing macrolide compound 11107D. The present invention relates to the following [1] to [15].

 [1]: Formula (I)

 11107B (i) Macrolide compound (hereinafter referred to as macrolide compound 11107B)

Formula (II)

16-hydroxylated macrolide compound (hereinafter referred to as macrolide compound)

DNA which is involved in the biological conversion to a protein having a hydroxylase activity at position 16 or ferredoxin, which partially or wholly encodes DNA or a variant thereof. Pure DNA isolated.

 [2]: The DNA according to [1], represented by the following (a), (b) or (c):

 (a) DNA encoding a protein having a hydroxylase activity at position 16 of macrolide compound 11107B, which is a continuous base sequence from base 1322 to base 2548 of SEQ ID NO: 1, a base of SEQ ID NO: 2 A DNA selected from the group consisting of a continuous base sequence from 420 to base 1604 and a continuous base sequence from base 172 to base 1383 of SEQ ID NO: 3.

 (b) a variant of the DNA shown in (a) above,

 (i) hybridizing with the DNA shown in (a) under stringent conditions, and

 (ii) DNA encoding a protein having a 16-position hydroxylase activity of macrolide compound 11107B.

 (c) does not hybridize with the DNA shown in (a) under stringent conditions due to degeneracy of gene codons, but is encoded by the DNA shown in (a) or (b) DNA that encodes a protein that has the same amino acid sequence as the protein.

 [3]: A protein encoded by the DNA according to [2].

 [4]: An autonomously replicating or integrated replicating recombinant plasmid carrying the DNA of [2].

 [5]: A transformant transformed with the recombinant plasmid of [4].

 [6]: A DN encoding a protein having a 16-position hydroxylase activity of macrolide compound 11107B, wherein the DNA described in [2] or a DNA comprising a part thereof is used as a probe or primer. A isolation method.

 [7]: The DNA according to [1], which is represented by the following (d), (e) or (f):

(d) F: DNA encoding t-redoxin, consisting of 2564 bases of SEQ ID NO: 1 From base 1643 to base 1834 of SEQ ID NO: 2 and a base sequence from base 1399 to base 1593 of SEQ ID NO: 3 DNA.

 (e) a variant of the DNA shown in (d) above,

 (i) hybridizing with the DNA shown in (d) under stringent conditions, and

 (ii) DNA encoding a protein having a ferredoxin function.

 (f) does not hybridize with the DNA shown in (d) under stringent conditions due to degeneracy of the gene codon, but is encoded by the DNA shown in (d) or (e) DNA encoding a protein having the same amino acid sequence as the protein.

 [8]: A protein encoded by the DNA according to [7].

 [9]: [7] An autonomously replicating or integrated replicating recombinant plasmid carrying the isolated DNA.

 [10]: A transformant transformed with the recombinant plasmid according to [9].

 [11]: A method for isolating a DNA encoding a protein having a ferredoxin function, comprising using the DNA described in [7] or a DNA comprising a part thereof as a probe or a primer.

 [12]: The transformant according to [5] or [10] is cultured in a medium, and grown during or after culturing,

[Where, W represents

R ¹² , R ^16b , R ^17a , R ^17b , R ¹⁸ , R ^20a , R ^20b , R ^21a and R ^{2 are the} same or different,

 (1) hydrogen atom,

(2) ₂₂ alkyl group which may have a substituent,

 (3) -OR (where R is

 • 1) hydrogen atom,

 Which may have a substituent,

2) C ₂₂ alkyl group,

3) C ₇ one ₂₂ Ararukiru group,

 4) 5- to 14-membered heteroaryloxyalkyl group,

5) C ₂ - ₂₂ Arukanoiru group,

6) C ₇ — ₁₅ Aloynole group,

7) C ₃ _ ₂₃ unsaturated alkanoyl group,

8) —COR ^c . (In the formula, Rc. May have a substituent,

 8-1) 5- to 14-membered heteroaryl group,

8-2) C ₂₂ alkoxy group,

8-3) unsaturated C ₂ - ₂₂ alkoxy group,

8-4) C ₆ _ ₄ aryloxy group,

 8-5) 5- or 14-membered heteroaryloxy group,

 Or

 8-6) 3 to 14-membered nitrogen-containing non-aromatic heterocyclic ring),

9) C Bok ₂₂ alkylsulfonyl group,

10) c ₆ — ₁₄ arylsulfonyl group

Or 2004/017906

1 1) One S i R ^sl R ^{s 2} R ^s3 (wherein, R ^sl , R ^{s 2} and R ^{s 3} are the same or different and represent a ァ alkyl group or a aryl group),

 (4) Halogen atom

Or

{Wherein, R ^M represents a single bond or 110 CO—;

R ^N1 and R ^N2 are

 1) same or different,

 1-1) hydrogen atom or

 • 1-2) may have a substituent,

ω ₂₂ alkyl group,

(ii) an unsaturated C ₂ _ ₂₂ alkyl group,

(iii) C ₂ over ₂₂ Arukanoiru group

(iv) C ₇ - ₁₅ Aroiru group,

(v) unsaturated C ₃ one ₂₃ Arukanoiru group,

(vi) 〇 ₆ _ ₁₄ reel group,

 (vii) a 5- to 14-membered heteroaryl group,

(viii) C ₇ _ ₂₂ Ararukiru group,

(ix) C— ₂₂ alkylsulfonyl group or

(X) represents a C ₆ _ ₁₄ arylsulfonyl group,

 Or

2) R ^N1 and R ^N2 together with the nitrogen atom to form a 3- or 14-membered nitrogen-containing non-aromatic heterocyclic ring which may have a substituent);

 However,

R ^{21 a} and R ^21b are together a connexion, (i) a ketone structure (= 0) or (ii) Okishi beam structure {= NOR. ^x (wherein, R. ^x may have a substituent, C - ₂₂ alkyl group, unsaturated C ₂ - ₂₂ alkyl groups, C ₆ - ₁₄ Ariru group, a 5-membered ring to 14-membered ring . _ heteroaryl group, or a C _{7 2} Ararukiru a group)} may be formed; R ^16a represents a hydrogen atom;

R ^{21 c}

 (1) hydrogen atom or

 (2)

(Wherein, R ^22a , R ^22b and R ^22c are the same or different,

 • 1) hydrogen atom,

2) Ci- ₆ alkyl group,

 3) —OR (where R has the meaning described above),

4) one R ^M — NR ^N1 R ^N2 (where R ^M , R ^N1 and R ^N2 have the above meanings) or

 5) Halogen atom

 Represents;

 Or

One of R ^21a and R ^21b and one of R ^22a and R ^22b together form a partial structure orR ^21b )

May be formed;

G ^m

(1) A group represented by the formula (GM-1) PT / JP2004 / 017906

{Where,

R ² and R ^{1 Q} is to display the same or different connexion, hydrogen atom or C i _ ₂₂ alkyl group;

R ^3a , R ^3b , R ^5a , R ^5b , R ^6a and R ^6b are the same or different,

 1) hydrogen atom,

 2) hydroxy group,.

 3) may have a substituent,

 3- -1) — alkyl group,

3- -2) ₂₂ alkoxy groups,

3- -3) C ₄ aryloxy group

 3- -4) 5- or 14-membered heteroaryloxy group,

3--5) C2 ₂₂ alkanoyloxy group,

3- -6) c ₇ — ₁₅ aryloxy group

3- -7) C ₃ - ₂₃ unsaturated alk Noi Ruo alkoxy group,

3- -8) -OCOR ^co (wherein, Rc has the above meaning)

3- -9) C 1- ₂₂ alkylsulfonyl O alkoxy group,

3-10) C _{6 14} arylsulfonyloxy group

 Or

3-11) -OS i R ^sl R ^s2 R ^{s 3} (where R ^sl , R ^{s 2} and R ^{s 3} have the above-mentioned meanings),

4) Halogen atom Or

5) represents one R ^M —NR ^N1 R ^N2 , wherein R ^M , R ^N1 and R ^N2 have the above-mentioned meanings;

 Or

R ^5a and R ^5b may be taken together to form a ketone structure (= ；);

R ^{6 a} and R ^{6 b} is MAY together form a connexion, spiro O carboxymethyl oxiranyl group or Ekisomechire emissions group; or,

R ^7a and R ^7b may be the same or different, a hydrogen atom or a 〇_R ^H (wherein, R ^H is a hydrogen atom, ₂₂ alkyl or C ₂ - represents a ₂₂ Arukanoiru group)), (2) ( GM-II)

(Wherein, R ² , R ^3a , R ^3b , R ^6a , R ^6b , R ^7a , and shaku ぴ! ^ Are equivalent to the definition of equation (GM-1)),

 (3) a group represented by the formula (GM-III)

(GM-ΙΠ)

(Wherein R ² , R ^5a , R ^5b , R ^6a , R ^6b , R ^7a , R ^7b and R ¹⁰ are as defined in the formula ( ^GM -e)),

 (4) Group represented by the formula (GM-IV)

(Wherein, R ² , R ^6a , R ^7a , R ^7b and R ¹⁰ are as defined in the formula (GM-1))

Or

 (5) group represented by the formula (GM-V)

(Wherein R ² R ^3a , R ^6a , R ^6b and R ¹⁰ are as defined in the formula (GM-I)).

 With a macrolide compound represented by the formula (IV)

^{(Wherein, w, R 12, R 16b} , R 17a, R 17b, R 20a, R 20b, R 2l a, R 21 b, R 21 c Oyopi & ^∞ is the definition as defined in formula (III) Represent)

A method for producing a 16-position hydroxylated macrolide compound, which comprises converting into a 16-position hydroxylated macrolide compound, and collecting the converted 16-position hydroxylated macrolide compound.

 [13]: The production method according to [12], wherein the transformant is the transformant according to [5] and has a DNA encoding furedoxin.

 [14]: Formula (III—a)

R ⁵ ′ represents a hydrogen atom or an acetyl group, R ⁶ ′ represents a hydrogen atom or a hydroxy group, and R ⁷ ′ represents a hydrogen atom or an acetyl group), a compound represented by the formula (IV-a)

(Where 906

^ 4,

^{W ', R 5', R} 6 ' and R ^7' is a method for producing [12], wherein the converting to the reduction compounds of formula (III-a) is defined synonymous).

[15]: in the conversion of the compound of the formula (III-a) into the compound of the formula (IV-a),

A compound wherein R ⁵ ′, R ⁶ ′ and R ⁷ ′ are hydrogen atoms,

 (2)

Is a single bond, w 'is

1 ⁵ 'Pop ¹⁶ ' is a hydrogen atom, R ⁷ 'is a compound having an acetyl group,

 (3)

HL eight

 5-4 is a single bond, W 'is

A compound wherein R ⁵ ′ and R ⁷ ′ are a hydrogen atom, and R ⁶ ′ is a hydroxy group,

 (Four)

H / Q H

 Is a single bond, w 'is y ^

A compound in which R ⁵ ′ is a hydrogen atom, R ⁶ ′ is a hydroxy group, and R ⁷ ′ is an acetinol group;

 (Five)

⁵ -4 is a single bond, A compound in which W is a double bond, R ⁵ ′, R ⁶ ′ and R ⁷ ′ are hydrogen atoms, (6) is a single bond,

A compound in which W ′ is a double bond, R ⁵ ′ and R ⁶ ′ are a hydrogen atom, and R ⁷ ′ is an acetyl group, (7)

5-4 is a single bond,

A compound in which W ′ is a double bond, R ⁵ ′ and R ⁷ ′ are hydrogen atoms, R ⁶ ′ is a hydroxy group,

 (8)

5-4 is a single bond,

A compound in which W ′ is a double bond, R ⁵ ′ is a hydrogen atom, R ⁶ ′ is a hydroxy group, and R ⁷ ′ is an acetyl group,

 (9)

H ゝ H

⁵ 4 is a double bond, W, is ¹ ,

A compound wherein R ⁵ ′ and R ⁷ ′ are a hydrogen atom, and R ⁶ ′ is a hydroxy group,

 (Ten)

Ή eight H

 5 4 is a double bond, W, is,

A compound in which R ⁵ ′ is a hydrogen atom, R ⁶ ′ is a hydroxy group, and R ⁷ ′ is an acetyl group, (11) 0 H

 5-4 is a single bond, W 'is

Compounds in which R ⁵ ′ is an acetyl group, R ⁶ ′ is a hydroxy group, R ⁷ ′ is a hydrogen atom, and

(12)

[14] The production method according to [14], wherein the compound is a compound selected from the group consisting of a compound in which R ⁵ ′ is an acetyl group, R ⁶ ′ is a hydroxy group, and R ⁷ ′ is an acetyl group.

 [16]: Use of the transformant according to [5] or [10] for producing a 16-hydroxylated macrolide compound.

 According to the present invention, it is possible to isolate a DNA encoding a protein or ferredoxin having a 16-position hydroxylase activity of macrolide compound 11107B, determine its nucleotide sequence, and further carry the DNA. A transformant transformed with the plasmid and the plasmid was prepared, and the transformant was used to efficiently produce a 16-hydroxylated chloride compound.

 Hereinafter, embodiments of the present invention will be described in detail.

A microorganism capable of converting macrolide compound 11107B to macrolide compound 11107D

 In the present invention, a protein or furedoxin having a 16-position hydroxylase activity is obtained from cells collected from a culture obtained by culturing a microorganism capable of converting macrolide compound 11107B to macrolide compound 11107D. The partially or wholly encoding DNA can be isolated and sequenced. Then, an autonomously replicating or integrating replicating recombinant plasmid carrying the DNA is constructed, and a transformant is prepared using the plasmid.

The transformant prepared in this manner is cultured in a medium, and during or after culturing, Let '

The cultured transformant is brought into contact with the macrolide compound represented by the formula (III) to convert it into the 16-hydroxyl macrolide compound represented by the formula (IV). The 16-position hydroxylated macrolide compound can be obtained by collecting the 16-position hydroxylated macrolide compound.

 As a microorganism having the ability to convert the macrolide compound 11107B into the macrolide compound 11107D, any microorganism having such an ability can be used regardless of species and strains. In each case, Streptomyces sp. (Streptomyces sp.) Mer-11107 and A-1544 strains isolated from soil and unidentified actinomycetes A-1560 strain can be mentioned.

 In addition, Streptomyces sp. Mer-11107 is referred to as FERM P-18144, Research Institute of Life Science and Industrial Technology, National Institute of Advanced Industrial Science and Technology, 1-3-1, Tsukuba-Higashi, Ibaraki, 305-8566, Japan on December 19, 2000. And 1 November 1 Tsukuba East, Ibaraki 305-8566, Japan, November 27, 2001 1 National Institute of Advanced Industrial Science and Technology (AIST), Patent Organism Depositary Center (IP0D) At FERM BP-7812. The A-1544 strain is referred to as FERM P-18943 as of July 23, 2002 1-1-1 Higashi, Tsukuba, Ibaraki, Japan 305-8566 Japan, National Institute of Advanced Industrial Science and Technology (AIST), Central No. 6 Deposited at the Center, and on July 30, 2003, 1-chome, Tsukuba-Higashi 1-chome, 305-8566, Japan 1 National Institute of Advanced Industrial Science and Technology (AIST), Patent Organism Depositary Center (IP0D) At FERM BP-8446. A-1560 strain was deposited as FERM P-19585 on November 13, 2003 at 1-3-1 Higashi, Tsukuba-shi, Ibaraki, 305-8566, Japan on November 13, 2003. International deposit at the National Institute of Advanced Industrial Science and Technology (AIST) and the Patent Organism Depositary Center (IP0D) at Tsukuba-Higashi 1-chome, Ibaraki 305-8566, Japan, August 19, 2004 Transferred to FERM BP-10102.

 The bacteriological properties of the above strains are as follows.

 [Bacteriological properties of Mer-11107 strain]

 (1) Form

 Spirales aerial hyphae elongate from the base hyphae. Mature aerial hyphae

15 Revised Paper (Rule 91) First, a spore chain consisting of about 10 to 20 cylindrical spores is formed. The size of the spores is about 0.7 X 1.0; um, and the surface of the spores is smooth, and there are no special organs such as spores, sclerotium, and flagella.

 (2) Growth conditions in various media

 The following shows the culture characteristics after culturing on various media at 28 ° C for 2 weeks. The description of the color tone is indicated by the name of the color of Tresner's color wheels and the sign in parentheses.

 1) East's malt agar medium

 Growth is good, aerial hyphae are formed on the surface, and gray spores (Light gray; can be seen.) The reverse side of the culture is Light melon yellow (3ea) No soluble pigment is produced.

 2) Oatmeal agar medium

 It grows moderately, with slight aerial hyphae on its surface and gray spores (Gray; g). The reverse side of the culture is Nude tan (4 gc) or Putty (1 1/2 ec). No soluble dye is produced.

 3) Starch 'inorganic salt agar medium

 It grows well, with aerial hyphae on its surface and gray spores (Gray; e). The back of the culture is Fawn (4ig) or Gray (g). No soluble dye is produced.

 4) Glycerin. Asparagine agar medium

 It grows well and has aerial hyphae on its surface, showing white spores (White; a). The back of the culture is Pearl pink (3ca). No soluble dye is produced.

 5) Peptone 'Yeast' iron agar medium

 It grows poorly and does not form aerial hyphae on its surface. The back of the culture is Light melon yellow (3ea). No soluble dye is produced.

 6) Tyrosine agar medium

 It grows well and has aerial hyphae on its surface, showing white spores (White; a). The back of the culture is Pearl pink (3ca). It does not produce amyolytic pigment.

(3) Assimilation of various carbon sources The growth status after 2 weeks of cultivation at 28 ° C with various carbon sources added to Preedham's Gottlieb agar medium is shown below.

 1) L-arabinose soil

 2) D-xylose ±

 3) D-glucose +

 4) D—Funolectose +

 5) Sucrose +

 6) Inositol +

 7) L—Ramnose I

 8) D—mannitol +

 9) Lahu North +

 (+ Assimilate, soil assimilate somewhat, one hardly assimilate.)

 (4) Physiological properties

 The physiological properties of this bacterium are as follows.

 (a) Growth temperature range (East's malt agar medium, culture for 2 weeks): 12 ° C to 37 ° C

(b) Optimal temperature range (East 'malt agar medium, culture for 2 weeks): 21 ° C ~ 33 ° C

(c) Liquefaction of gelatin (glucose 'peptone' gelatin medium): negative

 (d) Milk coagulation (Skim milk medium): negative

 (e) Peptone formation of milk (Skim milk medium): negative

 (f) Starch hydrolysis (starch, inorganic salt agar medium): positive

 (g) Production of melanin-like pigment (Peptone 'East' iron agar medium): Negative

 (Tyrosine medium): Negative

 (h) Production of hydrogen sulfide (Peptone 'Yeast' Iron Agar Medium): Negative

 (i) Nitrate reduction (broth containing 0.1% potassium nitrate): negative

 (j) Salt tolerance (yeast / malt agar medium, cultured for 2 weeks): grows with salt content of 4% or less

 (5) Cell components

LL-Diaminopimelic acid and glycine were detected in the cell wall of this bacterium. [Bacteriological properties of A-1544 strain]

 (1) Form

 Spira type aerial hyphae extend from the base hyphae. It forms a spore chain consisting of about 10 20 cylindrical spores in front of the mature aerial hyphae. The size of the spores is about 1.0 1.2 1.4 111, and the surface of the spores is spiny, and there are no special organs such as spores, sclerotium, and flagella.

 (2) Growth conditions in various media

 Table 1 shows the culture properties after culturing on various media at 28 ° C for about 2 weeks. Color notation is indicated by the color name of Tresner's color wheels and the sign in parentheses.

table 1

 (3) Assimilation of various carbon sources

Preed ham. Gottlieb agar medium with various carbon sources, 28 ° C, culture for 2 weeks Table 2 shows the subsequent growth status.

Table 2

 +: Assimilate, ±: assimilate somewhat, 1: assimilate hardly

 (4) Physiological properties

 The physiological properties of this bacterium are as follows.

 (a) Growth temperature range (Yeast / malt agar medium, culture for 2 weeks): 15 ° C to 41 ° C

 (b) Optimum growth temperature (Yeast / malt agar medium, culture for 2 weeks): 20 ° C to 37 ° C

 (c) Liquefaction of gelatin (glucose, peptone, gelatin medium): positive

 (d) Milk coagulation (skim milk medium): positive

 (e) Peptone formation of milk (Skim milk medium): Positive

 (f) Starch hydrolysis (starch, inorganic salt agar medium): positive

 (g) Melanin-like pigment production (Peptone yeast 'iron agar medium): positive

 (Tyrosine medium): Negative

 (h) Production of hydrogen sulfide (peptone yeast 'iron agar medium): positive

 (i) Nitrate reduction (broth containing 0.1% potassium nitrate): Negative

 (j) Tolerance of salt (yeast / malt agar medium, cultured for 2 weeks): Growing at a salt content of 7% or less

 (5) Cell components

 LL-type diaminopimelic acid was detected in the cell wall of this bacterium.

 DNA of the present invention

The present inventors have coded from the above microorganisms a DNA involved in hydroxylation at position 16 of a macrolide compound, that is, a DNA encoding a protein having a 16-hydroxylase activity and a protein having a ferredoxin function. The DNA to be isolated and its base The sequence was determined. Hereinafter, DNA encoding a protein having a 16-hydroxyl enzyme activity and DNA encoding a protein having a ferredoxin function are collectively referred to as “DNAJ associated with 16-hydroxylase.

 The DNA encoding the protein having the 16-position hydroxylase activity of the present invention is represented by the following (1-1), (1-2) or (3).

 (1-1) a continuous base sequence from base 1322 to base 2548 of SEQ ID NO: 1, a continuous base sequence from base 420 to base 1604 of SEQ ID NO: 2 and a base from base 172 to base 1383 of SEQ ID NO: 3 DNA selected from the group consisting of the nucleotide sequences described above.

 (1-2) a modified form of the DNA represented by the above (1-1),

 (i) a nucleotide sequence that hybridizes under stringent conditions with any of the DNAs set forth in (1-1) above, and

 (ii) DNA encoding a protein having a 16-hydroxyl enzyme activity of a macrolide compound.

 (1-3) Due to the degeneracy of gene codons, it does not hybridize under stringent conditions to any of the DNAs shown in (1-1) above, but does not hybridize with any of the above (1-1) or (1-2). DNA encoding a protein having the same amino acid sequence as the protein encoded by the indicated DNA.

 The “16-position hydroxylase activity” refers to hydroxylating the macrolide compound 11107B represented by the formula (I) at position 16 and converting it to the macrolide compound 11107D represented by the formula (II) Means enzyme activity.

 The DNA encoding the protein having a ferredoxin function of the present invention is represented by the following (2-1), (2-2) or (2-3).

 (2-1) DNA encoding ferredoxin, a continuous base sequence from base 2564 to base 2761 of SEQ ID NO: 1, a continuous base sequence from base 1643 to base 1834 of SEQ ID NO: 2, and SEQ ID NO: DNA selected from the group consisting of a continuous base sequence from base 3399 to base 1593 of 3;

 (2-2) a modified form of the DNA shown in (2-1),

(i) hybridizing with the DNA shown in (2-1) under stringent conditions, And,

 (ii) DNA encoding a protein having a ferredoxin function.

 (2-3) Due to the degeneracy of gene codons, it does not hybridize with the DNA shown in (2-1) under stringent conditions, but is not shown in (2-1) or (2-2). DNA encoding a protein having the same amino acid sequence as the protein encoded by the DNA.

 The “ferredoxin function” means a protein function that transfers electrons to the 16-hydroxylase and performs a hydroxylation reaction with the 16-hydroxylase.

 The term “base sequence that hybridizes under stringent conditions” in the above description of DNA refers to a colony hybridizer that uses any one of the aforementioned DNAs (1-1) or (2-1) as a probe. A DNA sequence obtained by using the method such as the seeding method, plaque hybridization method, or Southern plot hybridization method.For example, immobilization of colony or plaque-derived DNA or a fragment of the DNA. After performing hybridization at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filtered filter, 0.1 to 2 × SSC solution (1 × SSC solution is 150 raM sodium chloride, 15 mM DNA that can be identified by washing the filter 1 at 65 ° C with sodium citrate). The method described in Molecular Cloning: A laboratory Mannua II, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989 (hereinafter abbreviated as "Molekiyura 1. Cloning 2nd edition") It can be carried out according to.

 Examples of DNA that hybridizes under stringent conditions include DNA having a certain degree of homology with the base sequence of DNA used as a probe, for example, 80% or more, preferably 85% or more, and more preferably 90% or more. %, More preferably 95% or more, and most preferably 98% or more.

The method for obtaining the 16-position hydroxylase-related DNA of the present invention is not particularly limited. Based on the nucleotide sequence information set forth in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 in the sequence listing in this specification, appropriate probes and primers are prepared and used to belong to actinomycetes. The DNA of the present invention can be isolated by screening a DNA library of a microorganism to be used. The DNA library can be prepared by a conventional method from a microorganism expressing the 16-position hydroxylase activity.

 The 16-position hydroxylase-related DNA of the present invention can also be obtained by PCR. Using a DNA library derived from the microorganism described above as type I, PCR was performed using a pair of primers designed to amplify any of the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. Do. PCR reaction conditions can be set as appropriate. For example, a reaction consisting of 94 ° C for 30 seconds (denaturation), 55 ° C for 30 seconds to 1 minute (annealing), and 72 ° C for 2 minutes (extension) As one cycle of the process, for example, 30 cycles may be performed, followed by a reaction at 72 ° C. for 7 minutes. The amplified DNA fragment can then be cloned into a vector that can be amplified in a suitable host.

 Operations such as the preparation of the above-described probe or primer, construction of a DNA library, screening of a DNA library, and cloning of a target gene are known to those skilled in the art.For example, molecular cloning, 2nd edition, Current

Protocols in Molecular Biology, supplements 1 to 8, can be carried out according to the method described in John Wiley & Sons (1987-1997) and the like.

 The method for obtaining the protein of the present invention is not particularly limited, and may be a protein synthesized by chemical synthesis or a recombinant protein produced by a gene recombination technique. When producing a recombinant protein, first, a DNA encoding the protein described above in the present specification is obtained. The protein of the present invention can be produced by introducing this DNA into an appropriate expression system. The expression of the protein in the expression system will be described later in this specification.

The recombinant vector of the present invention

 The DNA of the present invention can be used by inserting it into an appropriate vector. The type of vector used in the present invention is not particularly limited. For example, an autonomously replicating vector

(Eg, a plasmid) or, when introduced into the host cell, is integrated into the host cell genome and replicated along with the integrated chromosome. JP2004 / 017906

Is also good. In the expression vector, the DNA of the present invention is operably linked to elements required for transcription (for example, a mouth motor). A promoter is a DNA sequence that exhibits transcriptional activity in a host cell, and can be appropriately selected according to the type of host. Transformant of the present invention and production of recombinant protein using the same

 A transformant can be prepared by introducing the DM or recombinant vector of the present invention into an appropriate host. The host cell into which the DNA or recombinant vector of the present invention is introduced may be any cell as long as it can express the gene of the present invention, and includes bacteria, yeast, fungi, and higher eukaryotic cells. Examples of bacterial cells include Gram-positive bacteria such as Bacillus or Streptomyces or Gram-negative bacteria such as Escherichia coli. Transformation of these bacteria may be carried out by using a competent cell by a protoplast method, an electoral poration method or other known methods. For example, the electroporation method can be performed as follows. The plasmid containing the foreign gene is added to the suspension of the competent cells, and the suspension is placed in a cuvette dedicated to electroporation, and a high-voltage electric pulse is applied to the cuvette. . Thereafter, the cells are cultured in a selection medium, and the transformant is isolated on a plate agar medium. Examples of yeast cells include cells belonging to Saccharomyces or Schizosaccharomyces, for example, Saccharomyces' Saccharomyces

cerevisiae) or Saccharomyces ^ Seth Kunorei to Li (S accharomyces kluyveri) Hitoshiryoku ^Λ and the like. Examples of a method for introducing a recombinant vector into a yeast host include an electrotrophic method, a spheroplast method, and a lithium acetate method. Examples of other fungal cells are filamentous fungi, such as cells belonging to Aspergillus, Neurosbora, Fusarium; or Trichoderma. When a filamentous fungus is used as a host cell, transformation can be performed by integrating the DNA construct into the host chromosome to obtain a recombinant host cell. Integration of the DNA construct into the host chromosome can be performed according to known methods, for example, by homologous recombination or heterologous recombination. The above transformant is cultured in an appropriate nutrient medium under conditions allowing expression of the introduced gene. In order to isolate and purify the protein of the present invention from the culture of the transformant, a conventional protein isolation and purification method may be used. For example, if the protein of the present invention is expressed in a lysed state in the cells, after the culture is completed, the cells are collected by centrifugation, suspended in an aqueous buffer, and then sonicated by an ultrasonic crusher or the like. Crush to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a normal protein isolation and purification method, that is, a solvent extraction method, a salting-out method with ammonium sulfate, a desalting method, a precipitation with an organic solvent, Method, anion exchange chromatography using a resin such as getylaminoethyl (DEAE) Sepharose, SP-

A cation exchange chromatography method using a resin such as Sepharose FF (manufactured by Amersham Biosciences), a hydrophobic chromatography method using a resin such as ptinoresepharose and pheninoresepharose, and a gel using a molecular sieve. A purified sample can be obtained using a single or a combination of techniques such as filtration, affinity chromatography, chromatography, and electrophoresis such as isoelectric focusing.

 Method for producing 16-position hydroxylated macrolide compound

 The present invention uses a transformant into which a DNA encoding a protein having a 16-position hydroxylase activity or a DNA encoding a protein having a ferredoxin function has been introduced, and in the presence of the transformant, the above-mentioned formula (III) A method for producing a 16-hydroxyl-hypermacrolide-based compound represented by the above formula (IV), comprising hydroxylating the macrolide-based compound represented by the formula (IV).

 The macrolide compound that can be hydroxylated by the transformant of the present invention is a macrolide compound represented by the above formula (ΙΠ) (a macrolide compound represented by the above formula (IV)), preferably A macrolide compound represented by the formula (ΠΙ-a)

(A macrolide compound represented by the above formula (IV-a)), and more preferably a macrolide compound 11107B (macrolide compound 11107D). The figures in parentheses are the hydroxylated macrolide compounds at the 16-position.

 The conditions for hydroxylating the macrolide compound in the presence of the transformant are as follows.

First, if necessary, an inducer is added to the 16-hydroxylase-related DNA in the transformant to express it in the cells. The bacterial cells expressing these DNAs are used as substrates for the above formula (III) And the conversion reaction. The temperature of the conversion reaction can be appropriately determined in consideration of the optimal temperature of the transformant. The reaction time can also be appropriately determined in consideration of the conversion rate to the 16-position hydroxylated macrolide compound (the degree of progress of the reaction) and the like. For example, 1 to 5 days at 20 to 31 ° C is suitable. Further, the reaction can be carried out in any of a batch mode and a continuous mode.

 For the isolation and purification of the produced 16-hydroxylated macrate compound, a separation and purification method generally used for isolating a microbial metabolite from its culture solution can be used. For example, organic solvent extraction using methanol, ethanol, acetone, butanol, ethyl acetate, butyl acetate, chloroform, toluene, etc., adsorption / desorption using a hydrophobic adsorption resin such as Diaion HP-20, SEPHADEX LH -Any known method such as gel filtration chromatography using -20, etc., adsorption chromatography using activated carbon, silica gel, etc., or adsorption / desorption treatment using thin-layer chromatography, or high-performance liquid chromatography using reversed phase chromatography, etc. This is the method. The method is not particularly limited to the method shown here. The desired 16-position hydroxylated macrolide compound can be isolated and purified by using these methods singly or in any order, and by repeatedly using them.

 In the present invention, the modified DNA means a DNA modified by deletion, conversion, addition, insertion or the like of a constituent base, or a derivative thereof, and a DNA exhibiting the same effect as the original DNA. Means '

 Further, the “halogen atom” used in the specification of the present application means a fluorine atom, a chlorine atom, a bromine atom ”, and an iodine atom.

Used herein as a "Ji _{2 2} alkyl group", it has from 1 to carbon atoms shows two two linear or branched alkyl group such as methyl group, Echiru group, n- flop port propyl group, IS 0-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2 —Dimethylpropyl group, 1-ethylpropyl group, n-hexyl group, 1-ethyl-2-methylpropyl group, 1,1,2—tri Methyl butyl group, 1-methylbutyl group, 2-methylbutyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 2,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2,3 —Dimethylbutyl group, 1-ethylbutyl group, 2-ethylbutyl group, 2-methylpentyl group, 3-methylpentyl group, n-heptyl group, n-octyl group, n-nonyl group, n-decyl group, etc. And preferably represents a linear or branched alkyl group having 1 to 6 carbon atoms, for example, a methyl group, an ethyl group,: n-propynole group, iso-propynole group, n-butyl group, iso- Butyl, sec-butynole, tert-butynole and the like.

The "unsaturated C ₂ _ _{2 2} alkyl group" used herein, 2 to carbon atoms 2 two linear or branched alkenyl group, or a carbon number of 2 to 2 two linear or Represents a branched alkynyl group, such as vinyl group, aryl group, 1-propenyl group, isopropenyl group, 2-methyl-1-propenyl group, 2-methyl-2-propenyl group, 1-butenyl Group, 2-ptenyl group, 3-pthenyl group, 1-pentenyl group, 1-hexenyl group, 1,3-hexanegenyl group, 1,5-hexanedienyl group, ethenyl group, 1-propynyl group, 2 —Propynyl group, 1-butynyl group, 2-butynyl group, 3-butynyl group, 1-ethyl-1 2-propynyl group, 2-methylinyl 2-propynyl group, 1-pentynyl group, 1- to Xinyl group, 1, 3 —Hexanediynyl group, 1, 5 — Xandiynyl group and the like, preferably a straight-chain or branched alkenyl group having 2 to 10 carbon atoms, or a straight-chain or branched alkynyl group having 2 to 10 carbon atoms, for example, a Bier group , Aryl, 1-propenyl, isopropenyl, ethynyl, 1-propynyl, 2-propynyl, .1-butynyl, 2-butynyl, 3-butynyl, etc. .

Used herein as a "C ₆ _ _{1 4} Ariru group", 6 to mean 1 4 aromatic configured with charcoal atom hydrocarbon cyclic group, for example a monocyclic group, bicyclic And condensed rings such as tricyclic groups. Examples include phenyl, indenyl, 1-naphthyl, 2-naphthyl, azulenyl, heptalenyl, indacenyl, acenaphthyl, fluorenyl, phenenyl, phenanthrenyl, anthracenyl and the like. , Preferably phenyl, 1-naphthyl, 2-naphthyl And the like.

 As used herein, the term "5- to 14-membered heteroaryl group" refers to a monocyclic or bicyclic ring containing at least one heteroatom selected from the group consisting of a nitrogen atom, a sulfur atom and an oxygen atom. Refers to a 5- or 14-membered aromatic heterocyclic group of the formula or tricyclic. Preferable examples include, as nitrogen-containing aromatic heterocyclic groups, for example, pyrrolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazuryl, triazolyl, tetrazolyl, benzotriazolyl, pyrazolyl, Imidazolyl group, benzimidazolyl group, indolinole group, isoindolyl group, indolizinyl group, prenyl group, indazolyl group, quinolinyl group, isoquinolinyl group, quinolizinyl group, phthalazinyl group, naphthyridinyl group, quinoxalinyl group, quinazolinyl group, quinazolinyl group, quinazolinyl group Group, imidazotriazinyl group, birazinopyridazinyl group, acridinyl group, phenanthridinyl group, carbazolyl group, sorbazolinyl group, perimidinyl group, phenantholinyl group, phenacinyl group, A midazopyridinyl group, an imidazopyrimidinyl group, a pyrazolipid pyridinyl group, a pyrazolipid pyridinyl group, etc .; examples of the sulfur-containing aromatic heterocyclic group include, for example, chenyl group, benzothenyl group, etc .; For example, a furyl group, a vinyl group, a cyclopentanyl group, a benzofuryl group, an isobenzofuryl group, etc .; Examples of an aromatic heterocyclic group containing two or more heterogeneous hetero atoms include, for example, a thiazolyl group, an isotizazo group. Ryl, benzothiazolyl, benzothiadiazolyl, phenothiazidyl, isoxazolyl, furazanyl, phenoxazinyl, oxazolyl, isoxazolyl, benzoxazolyl, oxaziazolyl, virazoloxazolyl, Thiazolyl group, Chenofuranyl group, Flopi Lil groups, pyridinium Do Kisajiniru group and the like, preferably thienyl group, furyl group, pyridinyl group, pyridazinyl group, pyrimidinyl group, Birajiniru group.

As used herein, the term “3- or 14-membered nitrogen-containing non-aromatic heterocycle” refers to a monocyclic, bicyclic or tricyclic 3- to 14-membered ring containing at least one nitrogen atom. Refers to a non-aromatic heterocycle. Preferred examples include, for example, aziridinyl, azetidinole, pyrrolidinyl, pyrrolyl, piperidinyl, piperazinyl, homopyridinyl 6

Examples include a lysinyl group, a homopiperazinyl group, an imidazolyl group, a birazolidinyl group, an imidazolidinyl group, a morpholinyl group, a thiomorpholinyl group, an imidazolinyl group, an oxazolinini / re group, and a quinutalidinyl group. The nitrogen-containing non-aromatic complex ring also includes a group derived from a pyridone ring and a non-aromatic condensed ring (for example, a group derived from a phthalimid ring, a succinimido ring, etc.). It is.

Used herein as a "c ₂ _ ₂₂ Arukanoiru group", "Ji defined above

In _ ₂₂ alkyl group "means the end is a carbonyl group group, e.g. Asechiru group, a propionyl group, Puchiriru group, iso- Puchiriru group, valeryl group, iso- valeryl group, Bibariru group, forces Puroiru group, Dekanoiru Group, lauroyl group, myristoyl group, palmitoyl group, stearoyl group, arachidoyl group and the like, and preferably an alkanoyl group having 2 to 6 carbon atoms, for example, acetyl group, propionyl group, butyryl group, iso-butyryl group And the like.

Used herein - the "C _{7 15} Aroiru group", "C ₆ _ ₁₄ Ariru group" defined above, in the "5-membered ring to Teroariru group to 14-membered ring" is a carbonyl group bound to the end A benzoyl group, a 11-naphthoyl group, a 2-naphthoyl group, a picolinoyl group, a nicotinoyl group, an isonicotinoyl group, a fluoryl group and the like.

The “C ₃ _ ₂₃ unsaturated alkanoyl group” used in the present specification means a group in which a carbonyl group is bonded to the terminal in the above-mentioned “unsaturated C ₂ _ ₂₂ alkyl group”, for example, an acryloyl group, Examples include a propioyl group, a crotonyl group, an iso-crotonyl group, an oleroyl group, a linolenoyl group, and the like, and preferably a C2 to C6 unsaturated alkanoyl group, such as an acryloyl group.

The "C ₇ _ ₂₂ Ararukiru group" as used herein, the definition of "Ji Bok ₂

In ₂ alkyl group ", a substituted moiety is the definition of" C ₆ - means a ₁₄ Ariru group 7 to be replaced by "to 22 amino configured group carbon atoms, downy For example specifically Njiru Group, phenyl group, 3-phenylpropyl group, 4-phenylbutyl group, 1-naphthylmethyl group, 2-naphthylmethyl group, and the like. 7 to 10 aralkyl groups, such as a benzyl group and a phenethyl group.

Used herein the term "Ji Bok ^ alkoxy group" defined above, "Ji ₂

_{In the} “ ₂ alkyl group”, it means a group having an oxygen atom bonded to its terminal. Examples of suitable groups include methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, and iso- -Butoxy, sec-butoxy, tert -butoxy, n-pentyloxy, iso-pentyloxy, sec-pentinoleoxy, n-hexyloxy, iso-hexyloxy, 1,1-dimethylpropoxy, 1,2-dimethylpropoxy group, 2,2-dimethylpropoxy group, 2-ethylpropoxy group, 1-ethyl-2-methylpropoxy group,

1.2-trimethylpropoxy group, 1,2,2-trimethylpropoxy group, 1,1-dimethylbutoxy group, 1,2-dimethylbutoxy group, 2,2-dimethylbutoxy group, 2,3-dimethylbutoxy group, Examples thereof include a 1,3-dimethylbutoxy group, a 2-ethylbutoxy group, a 1,3-dimethylbutoxy group, a 2-methylpentyloxy group, a 3-methylpentyloxy group, and a hexyloxy group.

Used herein, "unsaturated C ₂ - _{2 2} alkoxy group" is a, in the "unsaturated c ₂ _ _{2 2} alkyl group" defined above, and meaning taste group having an oxygen atom attached to its end, preferably Examples of suitable groups include vinyloxy, aryloxy, 1-propenyloxy, isopropenyloxy, 2-methyl-1-propenyloxy, 2-methyl-2-propenyloxy, 1-butenyloxy, 2 —Butenyloxy, 3-butenyloxy, 1-pentyloxy, 1-hexenyloxy,

1.3-hexanegeninoleoxy group, 1,5-hexanegeninoleoxy group, propargyloxy group, 2-butynyloxy group and the like.

Used herein - the "〇 _{6 1 4} Ariruokishi group", the above-defined - in the "C _{6 1 4} Ariru group" means a group having an oxygen atom attached to its end, specifically, for example phenoxy Group, indenyloxy group, 1-naphthyloxy group, 2-naphthyloxy group, azulenyloxy group, heptalenyloxy group, indenyloxy group, acenaphthyloxy group, fluorenyloxy group, phenalenylo Xy, phenanthrenyloxy, anthracenyloxy and the like. As used herein, the term "5- to 14-membered heteroaryloxy group" refers to the "5- to 14-membered heteroaryl group" defined by Mitsumi and has an oxygen atom at its terminal. And specifically includes, for example, a pyrrolyloxy group, a pyridinyloxy group, a pyridazi-loxy group, a pyrimidinyloxy group, a pyrazinyloxy group, a triazolyloxy group, a tetrazolyloxy group, a benzotriazolyloxy group. Xy, pyrazolyloxy, imidazolyloxy, benzimidazolyloxy, indolyloxy, isoindolyloxy, indolizinyloxy, prinyloxy, indazolyloxy, quinolinyloxy, isoquinolinyloxy , Quinolidinyloxy, phthaladuloxy, naphthyridinyloxy Si, quinoxalinyloxy, quinazolinyloxy, cinnolinyloxy, pteridinyloxy, imidazotriazinyloxy, birazinopyridazinyloxy, ataridinyloxy, phenanthrini Roxy, carbazolyloxy, rivazolinyloxy, perimidinyloxy, phenantophyllinoxy, phenacinyloxy, imidazopyridinyloxy, imidazopyridimidinyloxy, villa Zolopyridinyloxy group, pyrazolipid pyridinyloxy group, chenyloxy group, benzocheeroxy group, furyloxy group, vinyloxy group, cyclopentaviranyloxy group, benzofuryloxy group, isobenzofuryloxy group, thiazolyloxy group Group, isothiazolyloxy group, benzo Azolyloxy, benzothiadiazolyloxy, phenothiazinyloxy, isoxazolyloxy, frazanyloxy, phenoxazinyloxy, oxazolyl ^ "xy, isoxazolyloxy Group, benzoxazolyloxy group, oxadiazolyloxy group, virazoloxazolyloxy group, imidazothiazolyloxy group, chenofuranyloxy group, flopyrroloxy group, pyridoxazinyloxy group, etc. And a phenyloxy group, a furyloxy group, a pyridyloxy group, a pyridazyloxy group, a pyrimidyloxy group, and a virazyloxy group.

As used herein, "5-membered ring to 14-membered heteroaryl The term "kill group" refers to a group in which the above-mentioned C alkyl group is substituted by the above-mentioned "5- to 14-membered heteroaryloxy group".

Used herein - the "^ 2 ₂ alkylsulfonyl. Le group", sulfo the "〇 ₂₂ alkyl group" defined bound - means group, specifically, for example, methane Sno Reho group, Examples include an ethanesulfonyl group, an n-prononsulfonyl group, an iso-propanesulfonyl group, and the like.

It used herein the term "C ₄ § reel sulfonyl group", the above-defined - means "C _{6 14} Ariru group" bonded to sulfonyl group, specifically, for example, benzenesulfonyl group, 1-naphthalene Examples include a sulfonyl group and a 2-naphthalenesulfonyl group.

As used herein, the term " ₂₂ alkylsulfonyloxy group" refers to a group in which an oxygen atom is bonded to the terminal in the "Ci- ₂₂ alkylsulfonyl group" defined above, for example, methylsulfonyloxy. Examples thereof include a xy group, an ethylsulfonyloxy group, an n-propinolesnorephonyloxy group, and an iso-propylsulfonyloxy group.

 The substituent "may have a substituent" used in the specification of the present application is

(1) a halogen atom,

 (2) hydroxyl group,

 (3) thiol group,

 (4) nitro group,

 (5) a nitroso group,

 (6) a cyano group,

 (7) carboxyl group,

 (8) sulfonyloxy group,

 (9) an amino group,

(10) Ci— ₂₂ alkyl group

(Eg, methyl group, ethyl group, n-propyl group, iso-propyl group, n-butyl group, iso-butyl group, sec-butyl group, tert-butyl group, etc.), (1 1) Unsaturated C _{2 —22} alkyl group

 (For example, vinyl group, aryl group, 1-propenyl group, isopropenyl group, ethynyl group, 1-propynyl group, 2-propynyl group, 1-butynyl group, 2-butynyl group, 3-butynyl group etc) ,

(12) C ₆ — ₁₄ reel group

 (Eg, phenyl, 1-naphthyl, 2-naphthyl, etc.),

 (13) 5- or 14-membered heteroaryl group

 (For example, chenyl group, furyl group, pyridinyl group, pyridazinyl group, pyrimidinyl group, pyrazur group, etc.),

 (14) 3- or 14-membered nitrogen-containing non-aromatic heterocyclic ring

 (For example, aziridinyl group, azetidyl group, pyrrolidinyl group, pyrrolyl group, piperidinyl group, piperazinyl group, imidazolyl group, bilazolidinyl group, imidazolidinyl group, morpholinyl group, imidazolinyl group, oxazolinyl group, quinuclidil group, etc.)

(15) C - _{2 2} alkoxy group

 (For example, methoxy group, ethoxy group, n-propoxy group, iso-propoxy group, sec-propoxy group, II-butoxy group, iso-butoxy group, sec-butoxy group, tert-butoxy group, etc.),

(16) C ₆ — ₁₄ aryloxy group

 (For example, phenoxy group, 1-naphthyloxy group, 2-naphthyloxy group, etc.),

(1 7) C ₇ — ₂₂ aralkyloxy group

 (Eg, benzyloxy, phenethyloxy, 3-phenylpyroxy, 4-phenylbutyloxy, 1-naphthylmethyloxy, 2-naphthylmethyloxy, etc.)

 (18). 5- to 14-membered heteroaryloxy group

 (For example, chenyloxy group, furyloxy group, pyridinyloxy group, pyridazinyloxy group, pyrimidinyloxy group, virazinyloxy group, etc.),

(19) C ₂ — ₂₃ alkanoyl group (E.g., acetyl, propioyl, butyryl, iso-butyryl, valeryl, iso-valeryl, bivalyl, forceproyl, decanoyl, radioyl, myristoyl, palmitoyl, stearoyl Group, arachidoyl group, etc.)

(20) C ₇ — ₁₅ aryloyl group

 (For example, benzoyl group, 1-naphthoyl group, 2-naphthoyl group, etc.),

(21) C ₃ _ ₂₃ unsaturated Arukanoiru group

 (For example, atariloyl group, propioyl group, crotonoyl group, iso-crotonyl group, oleroyl group, linolenoyl group, etc.),

(22) C ₂ one ₂₃ Arca Noi Ruo alkoxy group

(E.g., Asetokishi group, a propionyloxy Ruo alkoxy group, Akuriruokishi group), (23) C ₂ one ₂₂ alkoxycarbonyl group

 (For example, methoxycarbonyl group, ethoxycarbonyl group, n-propoxycarbonyl group, is 0-propoxycanoleponyl group, n-butoxycanoleboninole group, is 0-butoxycarbonyl group, sec-butoxycarbonyl group, tert-hydroxycarbonyl group)

(24) unsaturated C ₃ - ₂₂ alkoxycarbonyl group

 (Vinyloxycarbonyl group, aryloxycarbonyl group, 1-propenyloxycarbonyl group, isopropininoleoxycarbonyl group, propargyloxycarbonyl group, 2-butynyloxycarbonyl group),

(25) C, ₂₂ alkylsulfonyl group

 (For example, a methanesulfonyl group, an ethanesulfonyl group, an n-propanesulfonyl group, an is0-propanesnolephonyl group, etc.),

(26) C ₆ — ₁₄ arylsulfonyl group

 (For example, benzenesulfonyl group, 1-naphthalenesulfonyl group, 2-naphthalenesulfonolene group, etc.) and

(27) C ^ ₂₂ alkylsulfonyloxy group

(For example, methanesulfonyloxy group, ethanesulfonyloxy group, n-propa 04 017906

Sulfonyloxy group, iS0-propanesulfonyloxy group, etc.)

And a group selected from the group consisting of Example

 Reference Example 1 (Manufacture of Mac-Moleride Compound 11107B as Raw Material)

 From a slant culture (ISP-2 medium) of Streptomyces sp. (Streptomyces sp.) Mer-11107 strain (FERM BP-7812), 1 loop of platinum loop was added to 50 ml of seed culture medium [glucose 2%, ES SANMEET (manufactured by Ajinomoto Co., Inc.). 1%, 0.5% yeast extract (manufactured by Oriental Yeast Co., Ltd.), 0.25% sodium salt, 0.32% calcium carbonate, pH 6.8 before sterilization. The flask was inoculated and cultured at 28 ° C for 2 days to obtain a first stage seed culture. 0.1 ml of this culture was inoculated into a 500 ml triangular flask containing 100 ml of the same seed culture medium, and cultured at 28 ° C. for 1 day to obtain a second stage seed culture. 800 ml of the second-stage seed culture obtained in this manner was used in a production medium [soluble starch 5%, pharmamedia 0.8 °, gluten meal 0.8%, yeast extract 0.5%, calcium carbonate 0.1 %, PH 6.8 before sterilization] Inoculate into a 200 L tank containing 100 L, culture at 28 ° C with agitation of 90 rpm, aeration rate 1. Ovvm, aeration and agitation culture for 5 days at an internal pressure of 20 kPa A liquid was obtained.

After extracting a part (10 L) of the culture solution thus obtained with 10 L of 1-butanol, the butanol layer was dried under reduced pressure to obtain 100 g of a crude active fraction. This crude active fraction was added onto SEFFADEX LH-20 (Pharmacia, 1500 ml) and eluted with a solvent of tetrahydrofuran-methanol (1: 1). The fraction eluted from 540 ml to 660 ml was concentrated to dryness under reduced pressure to obtain a residue (660 mg). The residue was dissolved in a mixture of ethyl acetate and methanol (9: 1; v / v), and subjected to silica gel column chromatography (Kogel 0200, 50 g). The column was eluted with a mixture (2 L) of n-hexane and ethyl acetate (l: 9; v / v). The fractions eluted from 468 ml to 1260 ml were collected and concentrated under reduced pressure. 25 _{mg of a} crude active fraction was obtained. .

The obtained crude active fraction was subjected to preparative high-performance liquid chromatography (HPLC) under the following HPLC preparative conditions (A), fractions eluted at a retention time of 34 minutes were collected, and acetonitrile was distilled. After the separation, the fraction was subjected to desalting by HPLC under the following HPLC preparative conditions (B). 7906

6 mg of a macrolide compound 11107B (retention time: 37 minutes) was obtained.

 HPLC preparative conditions (A)

Column: YMC-PACK ODS-A SH-343-5AM, 膽 20 brilliant X 250 mm (manufactured by Yemushi Corporation) Temperature: room temperature

Flow rate: 10ml / min

Detection: 240nm

Eluent: acetonitrile / 0.15% dihydrogen phosphate phosphate (pH 3.5) (2: 8-8: 2, v / v, 0-50 minutes, linear gradient)

 HPLC preparative conditions (B)

Column: YMC-PACK ODS-AM SH-343-5AM, φ20mm X 250mm (ヮ Emushi Corporation)

Flow rate: 10ml / min

Detection: 240nm '

Eluent: methanol / water (2: 8 to 10: 0, v / v, 0 to 40 minutes, linear gradient).

 Example 1: Determination of the nucleotide sequence of a gene derived from Streptomyces sp. A-1544 strain (FERM BP-8446)

 (1) Preparation of chromosomal DNA from Streptomyces sp. A-1544 strain

 A-1544 strain was inoculated into a medium consisting of glucose 1%, malt extract 0.4%, and yeast extract 1%, and cultured at 28 ° C for 3 days. The obtained culture was centrifuged at 3000 rpm for 10 minutes to collect the cells. Chromosomal DNA was prepared from the cells using a Blood & Cell Culture kit (QIAGEN).

 (2) Cloning of partial sequence of DNA encoding protein having hydroxylation activity at position 16 of macrolide compound 11107

 Streptomyces' Sericolor 3 (2) cytochrome? A mix 'primer (5Dm-3F (SEQ ID NO: 4) and 5Dm-3R (SEQ ID NO: 5)) was designed and prepared with reference to the amino acid sequence estimated to be 450 (¥? 10505).

A mixed base S (= C + G), Y (= C + T) was used to increase the reactivity in consideration of codon fluctuation. Next, a PCR reaction was performed using the two primers (5Dm-3F and 5Dm-3R) and the chromosomal DNA of the A-1544 strain obtained in (1) above as a template. The PCR reaction was performed using Takara LA Taq (Takara Shusha) and a PCR amplifier (Biometra T Gradient), denaturation at 98 ° C for 20 seconds, annealing at 50 ° C for 2 minutes, and elongation at 68 ° C for 30 minutes. A three-step reaction of 35 seconds was repeated 35 seconds. As a result, a DNA fragment having a size of about 500 bp (hereinafter referred to as DNA fragment-A1) was amplified. This DNA fragment-A1 is likely to be a part of DNA encoding a protein having hydroxylic activity. The DNA fragment-A1 amplified by the PCR reaction was recovered from the reaction solution by SUPREC PCR (Takara Shuzo).

 Next, in order to obtain a sufficient amount of DNA fragment-A1 to analyze the nucleotide sequence of the obtained DNA fragment-A1, DNA ligation kit ver. 2 (Takara Shuzo) was added to plasmid vector pT7Blue T (Novagen). Was used to ligate the DNA fragment-A1, and Escherichia coli JM109 strain was transformed. Then, ampicillin (50 IX g / mL), X-gal (5-bromo-4-chlofo-3-indoly β-D-galactoside; 40 β g / raL) ^ IPTG Usopropyl-β-D-thiogalactopyranoside; 100 Transformed Escherichia coli was selected using an L-Broth agar medium (1.0% bactotryptone, 0.5% yeast extract, 0.5% NaCl, 1.5% agar) containing Μ). The colonies of the transformed Escherichia coli thus isolated were cultured in an L-Broth liquid medium (1% pactotryptone, 0.5% yeast extract, 0.5% NaCl) containing ampicillin (50 g / mL). Plasmid DNA was separated and purified from the transformed transformed E. coli cells using a plasmid purification kit (QIAfilter Plasmid Midi Kit, QIAGEN) to obtain a fixed amount of DNA fragment-A1.

 (3) Analysis of nucleotide sequence of cloned DNA fragment-A1

 The nucleotide sequence of the DNA fragment-A1 obtained in (2) above was analyzed using a DNA nucleotide sequence analyzer (PE

Using Biosystems 377XL), the analysis was performed by the die terminator one cycle cycle method. As a result of the nucleotide sequence analysis, the DNA fragment-A1 amplified by the PCR reaction was measured to be about 500 bp by electrophoresis, but the nucleotide sequence analysis revealed that it was exactly 528 bp (SEQ ID NO: Base 1 from 1775 to base 2302). At both ends of the cloned 528 bp DNA sequence, DNA sequences corresponding to the two types of primers used in the above PCR reaction were found. Kind of It was revealed that the DNA was specifically amplified by the Braymers (5Dm-3F and 5Dm-3R).

 (4) Analysis of the region around DNA fragment-M

As described above, since the partial sequence of the DNA encoding the protein having hydroxylic activity derived from the A-1544 strain was determined, it was determined by inverse PCR (Cell Engineering Vol. 14, p. 591-593, 1995). The base sequence of the peripheral region extending upstream and downstream of the Clawing fragment was amplified, cloned, and sequenced. 'That is, the chromosomal DNA of the A-1544 strain (see (1)) was digested with restriction enzymes Pstl and Sail in H buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl ₂ , 10 mM diteositol, 100 mM NaCl), respectively. Digested. Each of the obtained restriction enzyme-cleaved DNA fragments was subjected to self-circulation using DNA Ligation Kit ver. 2 (Takara Shuzo).

 On the other hand, primers (6PIN-2F (SEQ ID NO: 6) and 6PIN-2R (SEQ ID NO: 7)) were designed and prepared from the base sequence of DNA fragment-A1.

 Next, PCR was carried out using these two primers (6PIN-2F and 6PIN-2.R) and the self-circularized A-1544 strain chromosomal DNA as a template. The PCR reaction was performed using Takara LA Taq (Takara Shuzo) and a PCR amplifying device (Biometra T Gradient). The two-step reaction was performed in which denaturation was performed at 98 ° C for 20 seconds and annealing and extension were performed at 68 ° C for 5 minutes for 35 minutes. Repeated several times.

As a result, a DNA fragment (DNA fragment-B1) with a size of about 3.5 kbp and a DNA fragment (DNA fragment-C1) with a size of about 2.8 kbp were amplified. co one de to DNA and DNA is a potential force ^{§ ι¾} · Rere having the DNA sequence containing the upstream and downstream regions.

DNA fragment-B1 and DNA fragment-C1 were recovered from this PCR amplification reaction solution by SUPREC PCR (Takara Shuzo). Next, in order to obtain a sufficient amount of each DNA fragment for the obtained DNA fragment-B1 and DNA fragment-C1 to analyze the base sequence, the plasmid vector pT7Blue T (Novagen ), DNA Ligation kit ver. 2 (Takara Shuzo), E. coli JM109 strain, and a plasmid purification kit (QIAfilter Plasmid Midi Kit, QIAGEN) to obtain a certain amount of each DNA fragment. 6

(5) Analysis of base sequence of DNA fragment-Bl (about 3.5 kbp size) and DNA fragment-C1 (about 2.8 kbp size)

 The nucleotide sequences of DNA fragment-B1 and DNA fragment-C1 obtained in (4) above were analyzed using a DNA sequence analyzer (PE Biosystems 377XL) according to the dye terminator-'cycle 'sequence method. The nucleotide sequence was analyzed, and information on the 3793 bp nucleotide sequence shown in SEQ ID NO: 1 was obtained from the DNA fragment-B1 and DNA fragment-C1 sequences.

 A search for the open 'reading frame (0RF) in the 3793 bp revealed that two proteins were encoded. A BLAST search of the amino acid sequences of these proteins revealed that 0RF (hereinafter, psmA) encodes a protein consisting of 409 amino acids with high homology to cytochrome P450 from base 1322 to base 2548 of SEQ ID NO: 1. There was). PsmA is most likely to be found in the amino acid sequence estimated to be cytochrome P450 (CYP105D5) in Streptomyces-cericala A3 (2) and the amino acid sequence estimated to be cytochrome P450 (CYP105D4) in Streptomyces lividans. It had high homology (72.6% homology) and relatively high homology to the cytochrome P450soy (SoyC) of Streptomyces griseus (Homology)

69.4%). This suggests that psmA may be a gene encoding a cytochrome P450-type hydroxylase and that 1 "is likely to be produced.

Immediately downstream of psmA (base 2564 to base 2761 of SEQ ID NO: 1), 0RF (hereinafter, referred to as psmB) encoding a protein having high homology to 3F-4S type ferredoxin was present. The protein encoded by psmB consists of 66 amino acids, and is a cytochrome of Streptomyces. Ferredoxin immediately downstream of the putative amino acid sequence of 450 (? 10505) has the highest homology to the putative amino acid sequence (83.3%), and the ferredoxin of Streptomyces griseus 307 ( 3 (^ 8) also had a relatively high ぃ homology (homology of 57.6 ° / ₀ ), so psmB encodes ferredoxin, which is responsible for electron transport and hydroxylates with psmA. It was thought that there was.

 Example 2: Construction of a transformant having psmA and psmB

(1) Preparation of DNA fragment containing both psmA and psmB from A-1544 strain T JP2004 / 017906

Referring to the nucleotide sequence of SEQ ID NO: 1 analyzed in Example 1, a primer DM-NdeF (SEQ ID NO: 8) having an Ndel site added at the 5 ′ end and a primer DM-NDM having an Spel site added at the 5 ′ end SpeR (SEQ ID NO: 9) was designed and created. Next, a PCR reaction was performed using these two primers (DM-NdeF and DM-SpeR) and the chromosomal DNA of the A-1544 strain obtained in Example 1 (1) as a template. PCR reaction is Takara LA

Using a Taq (Takara Shuzo) and a PCR amplifier (Biometra T Gradient), a two-step reaction in which denaturation was performed at 98 ° C for 20 seconds and annealing and extension were performed at 68 ° C for 2 minutes was repeated 30 times.

 As a result, a DNA fragment of about 1.5 kbp including psmA and psmB (hereinafter referred to as DNA fragment-D1) was widened. DNA fragment -D1 was recovered from this PCR amplification reaction solution by SUPREC PCR (Takara Shuzo).

 (2) Construction of plasmid pTC-DM

 pT7NS-CamAB (see W003 / 087381) in H buffer (50 mM Tris-HCl, pH 7.5, 10 mM

It was digested with restriction enzymes Ndel and Spel in MgCl ₂ , lOmM dithiothreitol, lOOmM NaCl) to obtain a plasmid digest. Similarly, the DNA fragment-D1 obtained in the previous section (1) is digested with restriction enzymes Ndel and Spel, and the obtained DNA fragment-D1 digest and plasmid digest are combined with DNA Ligation Kit ver. 2 (Takara Shuzo) Were connected. Yotsute thereto, DNA fragments containing therein both psmA and PSMB - and D1, plasmid pT7NS - (referred to as plasmid _P TC- DM) about the CamAB are connected 9. size 5kbp plasmid is It was constructed.

 (3) Preparation of E. coli transformant BL21 (DE3) / pTC-DM

 Escherichia coli BL21 (DE3) competent cells (Novagen) were transformed with the plasmid pTC-DM prepared in (2) above. Thus, E. coli BL21 (DE3) / pTC-DM strain transformed with the plasmid pTODM was obtained.

Example 3: Conversion of ME-265 represented by the following formula into ME-282 by a transformant of Escherichia coli having psmA and psmB

 ME-265

 ME-282

 (1) Preparation of transformant reaction solution

Transformed E. coli BL21 obtained in Example 2 (3) (DE3) / P TC- DM strain Contact Yopi

BL21 (DE3) / pT7NS- CamAB strain frozen seeds containing 3 mL of L-Broth medium (1.0% bactotryptone, 0.5% yeast extract, 0.5% NaCl) containing 50 μg / mL ampicillin The cells were inoculated into a 15 mL test tube and shake-cultured at 37 ° C for 20 hours. Add 500 μL of this seed culture to a 250 mL volume of 50 mL of L-Broth medium (1.0% batatotryptone, 0.5% yeast extract, 0.5% NaCl) containing 50 g / mL of ampicillin. Inoculate Erlenmeyer flask and shake culture at 32 ° C for 3 hours, then add lOOmM IPTG (isopropyl-β-D-thiogalactopyranoside) ¾r50μ 80mg / mL 5-amino levulinic acid 50βL sequentially, 32 ° C For 6 hours with shaking. The obtained culture was centrifuged (5000 rpm, 10 minutes) to collect the cells. This was suspended in 1.75 mL of 100 mM phosphate buffer (pH 6.1), and 80% glycerol was added at 250 mg / mL ME-265 at 50 / _tL. The conversion reaction solution thus obtained was reacted at 28 ° C. for 24 hours. The reaction mixture was extracted with 200 mL of acetonitrile, and the amounts of ME-265 and ME-282 were measured by HPLC. Table 3 shows the measurement results.

The detailed conditions of HPLC are shown below. PT / JP2004 / 017906 Analyzer: Shimadzu HPLC ΙΟΑνρ

Column: CAPCELL PAK C18 SG120 (φ4.6mm x 250mm)

Mobile phase: 45% acetonitrile (0-15 minutes)

 60% acetonitrile (15-30 minutes)

 45% acetonitrile (30-45 minutes)

Flow rate: lmL / min

Detection: UV240nm

Injection capacity:

Column temperature: 40 ° C

Analysis time: 45 minutes

Retention time: ME-265 24.8 minutes

 ME-282 12.7 minutes

Table 3

 (2) Obtaining ME-282 from the transformant reaction solution.

 4 mL of water was added to 1.8 mL of the reaction solution reacted for 24 hours, and extracted once with 8 mL of ethyl acetate and twice with 4 mL. The ethyl acetate layers were combined, dried over anhydrous sodium sulfate, and the solvent was removed. The obtained residue was purified by thin-layer chromatography (MERCK Silicagel 60 F254 0.25 mm developing solution; hexane: ethyl acetate = 1: 2) to obtain 0.2 mg of ME-282.

-丽R spectrum _{(CD 3 0D, 500MHz):} δ ppm ( integration, multiplicity, coupling constant J (Hz)):

0.87 (3H, d, J = 7.0Hz), 0.90 (3H, d, J = 7.0Hz), 0.94 (3H, t, J = 7.3Hz),

0.97 (3H d, J = 6.6Hz), 1.21-1.26 (1H, m), 1.29-1.37 (3H, m), 1.34 (3H, s), 1.44-1.52 (2H, m), 1.60-1.64 (1H , m), 1.65 (1H, dd, J = 6.2, 13.9Hz),

1.77 (3H, d, J = l.1Hz), 1.86 (1H, dd, J = 5.4, 13.9Hz), 1.89-1.94 (1H, m),

2.00 (3H, s), 2.43 (1H, dd, J = 5.5,13.9Hz), 2.50—2.60 (1H, m), 2.56 (1H, dd, J = 3.3, 13.9Hz), 2.66 (1H, dd, J = 2.2, 7.7Hz),

 2.89 (1H, dt, J = 2.2, 6.2 Hz), 3.52 (1H, dt, J = 4.8, 8.4Hz), 3.75-3.80 (1H, m) ,

4.90 (1H, overlapped with D ₂₀ ), 5.01 (1H, d, J = 10.6 Hz),

 5.42 (1H, dd, J = 9.2, 15.0 Hz), 6.13 (1H, d, J = 10.6 Hz), 6.52 (1H, dd, J = ll. 0, 15 0Hz). As a result, in the control strain E. coli BL21 (DE3) r7 ^ "311 ^ 8 strain, a peak which was regarded as ¾ © -282 was not obtained, but psmA and psmB were included.

In the BL21 (DE3) / pTC-DM strain, almost all of ME-265 was consumed, and a peak which appeared to be ME-282 was obtained. This suggests that psmA and psmB are involved in the conversion of Ε-265 to ME-282.

 Example 4: Conversion of macrolide-based compound 11107B to macrolide-based compound 11107D by Escherichia coli transformant having psmA and psmB

 (1) Preparation of transformant reaction solution

 As in Example 3, a test was performed using the macrolide compound 11107B as a substrate. The frozen seeds of the transformed bacteria BL21 (DE3) / pTC-DM strain and BL21 (DE3) / pT7NS-CamAB strain obtained in Example 2 (3) containing ampicillin 50 / g / mL were used. -Inoculated into a 15 mL test tube containing 3 mL of Broth medium (1.0% bactotryptone, 0.5% yeast extract, 0.5% NaCl), and shaken at 30 ° C for 20 hours ± nourishment. A 500 mL L-broth medium (50% ampicillin, 1.0% batatotryptone, 0.5% yeast extract, 0.5% NaCl) containing 50 g / mL of this seed culture was added to a 250 mL volume. After inoculating in an Erlenmeyer flask and culturing with shaking at 28 ° C for 5 hours, lOOraM IPTG usopropyl-] 3-D-thiogalactopyranoside) μ The cells were cultured with shaking at 25 ° C for 20 hours. The resulting culture was centrifuged (5000 rpm, 10 minutes) to collect the cells. Add this to lOOmM phosphate buffer

_(P H6. L) 1. was suspended in 75 mL, it was this 80% glycerol 250 mu L, a 40 mg / mL 11107B was added 50 μ ί. The conversion reaction solution thus obtained was reacted at 28 ° C. for 24 hours. The reaction solution (200 / iL) was extracted with 1 mL of acetonitrile, and the amounts of the macrolide compounds 11107B and 11107D were measured by HPLC. Table 4 shows the measurement results. The detailed conditions of HPLC are shown below.

Analyzer: Shimadzu HPLC ΙΟΑνρ Column: CAPCELL PAK C18 SG120 (φ4.6mm X 250mm)

 Mobile phase: 35% acetonitrile (0-10 minutes)

 35% to 65% acetonitrile (10 to 12 minutes)

 65% acetonitrile (12-15 minutes)

 35% acetonitrile (15-20 minutes)

 Flow rate: lmL / min

 Detection: UV240nm

 Injection capacity: IO L

 Column temperature: 40 ° C

 Analysis time: 20 minutes

 Retention time: 11107B 14.3 minutes

 11107D 7.9 minutes

Table 4

 (2) Acquisition of macrolide compound 11107D from reaction solution of transformant

 4 mL of water was added to 1.8 mL of the reaction solution reacted for 24 hours, and extracted once with 8 mL of ethyl acetate and twice with 4 mL. The ethyl acetate layers were combined, dried over anhydrous sodium sulfate, and the solvent was removed. The obtained residue was purified by thin-layer chromatography (MERCK Silicagel 60 F254 0.25 mm developing solution; ethyl acetate) to obtain 0.107 mg of 11107D.

- negation R spectrum _{(CD 3 0D, 500MHz):} δ ppm ( integration, multiplicity, coupling constant J (Hz)): 0. 87 (. 3H, d, J = 7 0Hz), 0. 88 ( 3H, d, J = 7.0 Hz), 0.93 (3H, t, J = 7.0 Hz), 1.18 (3H, s), 148-1.69 (8H, m), 1.33 ( 3H, s), 1.77 (3H, d, J = l. 1Hz), 1.82—1.90 (1H, m), 2.05 (3H, s), 2.49-2.60 ( 3H, m), 2.66 (1H, dd, J = 2.2, 8.2 Hz),

2.89 (1H, dt, J = 2.4, 5.7Hz), 3.52 (1H, dt, J = 4.8, 8.3Hz), 3.73—3.82 (1H, m) , 5.04 (1H, d, J = 9.8Hz), 5.05 (1H, d, J = 10.6Hz), 5.56 (1H, dd, J = 9.8, 15.2Hz), T / JP2004 / 017906

5.70 (1H, dd, J = 9.8, 15.2Hz), 5.86 (1H, d, J = 15.2Hz), 6.3 (1H, d, J = 10.8Hz),

 6.52 (1H, dd, J = 10.8, 15.2Hz).

As a result, in the control strain Escherichia coli BL21 (DE3) / pT7NS-CamAB strain, a peak which was considered to be a macrolide compound 11107D was not obtained, whereas the BL21 (DE3) / _P containing psmA and psmB was not obtained. In the TC-DM strain, a peak that appeared to be a macrolide compound 11107D was obtained. This suggests that psmA and psmB are involved in the conversion of macrolide compound 11107B to 11107D.

 Example 5: Conversion test on A-1544 self-closing mouth strain

 (1) Preparation of DNA fragment containing both psmA and psmB from A-1544 strain (for self-cloning)

 Referring to the nucleotide sequence of SEQ ID NO: 1 analyzed in Example 1, a primer DM-BglF (SEQ ID NO: 10) having a Bglll site added to the 5 ′ end and a primer DM-BglR having a Bglll site added to the 5 ′ end (SEQ ID NO: 11) was designed and created. Next, a PCR reaction was performed using these two primers (DM-BglF and DM-BglR) and the chromosomal DNA of the A-1544 strain obtained in Example 1 (1) as a template. The PCR reaction was performed using Takara LA Taq (Takara Shuzo) and a PCR amplification device (Biometra T Gradient) at 98 ° C for 20 seconds for denaturation, 63 ° C for 30 seconds for annealing, and 68 ° C for 4 minutes for elongation. The three-step reaction was repeated 30 times.

 As a result, a DNA fragment of about 3.5 kbp including psmA and psmB (hereinafter, referred to as DNA fragment-E1) was amplified. This PCR amplification reaction solution was subjected to agarose gel electrophoresis to fractionate. The above-mentioned DNA fragment-E1 having a size of about 3.5 kbp was cut out from the agarose gel and recovered by SUPREC 01 (Takara Shuzo).

 (2) Construction of plasmid pIJDMG

PIJ702 was digested with the restriction enzyme Bglll in H buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl ₂ , 10 mM dithiositol, 100 NaCl) to obtain a plasmid digest. Similarly, the DNA fragment-E1 obtained in (1) above is digested with the restriction enzyme Bglll, and the obtained DNA fragment-E1 digest and the plasmid digest are digested with DNA Ligation Kit ver. 2 (Takara Shuzo). And connected. As a result, a DNA fragment containing both psmA and psmB-E1, A plasmid (called plasmid pIJDMG) having a size of about 8.5 kbp linked to rasmid pIJ702 was constructed.

 (3) Preparation of self-cloning strain Α-1544 / pIJDMG

 Using the plasmid pIJDMG prepared in (2) above, the A-1544 strain was

Manipulation of Streptomyces -'A Laboratory Mamia 丄 John 丄 nnes

Transformation was performed according to the method described in Foundation, Norwich, 1985. Thus, the Α-1544 / pIJDMG strain transformed with the plasmid pIJDMG was obtained.

 Example 6: Conversion of 11107B to 11107D by self-cloning strain

Transformants were obtained in Example 5 (3) A- 1544 / pIJDMG strain, A- 1544 / pIJ702 strain, and frozen species mother original A- 1544 strains, SMN containing Chio thiostrepton 25 mu _g / mL Medium (Stabilose 2%, glucose 2%, essanmeat 2%, yeast extract 0.5%, NaCl 0.25%, CaC03 0.32% pH 7.4) Inoculated in a 250 mL Erlenmeyer flask containing 50 mL The cells were cultured with shaking at 28 ° C for 48 hours (seed culture, except that A-1544 strain was not supplemented with thiostrepton :). 0.5 mL of the obtained seed culture was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of SMN medium containing 25 μg / mL of thiostrepton, and cultured with shaking at 28 ° C for 72 hours (however, A- 1544 strain does not contain thiostrepton). 2 mL of the obtained culture was dispensed, and 100 L of 1 M phosphate buffer (pH 6.5) and 50 L of 100 rag / mL 11107B were added thereto. The converted culture solution thus obtained was reacted at 28 ° C for 12 hours. The reaction solution was extracted with 1 mL of acetonitrile, and the amounts of 11107B and 11107D were measured by HPLC. Table 5 shows the measurement results. The detailed conditions of HPLC are shown below.

 Analyzer: Shimadzu HPLC ΙΟΑνρ

 Column: CAPCELL PAK C18 SG120 ((i) 4.6mraX 250mm)

 Mobile phase: 35% acetonitrile (0-10 minutes)

 35% to 65% acetonitrile (10 to 12 minutes)

 65% acetonitrile (12-15 minutes)

 35% acetonitrile (15-20 minutes)

 Flow rate: lmL / min

Detection: UV240nm PT / JP2004 / 017906

Injection capacity: IO I L

 Force ram temperature: 40 ° C

 Analysis time: 20 minutes

 Retention time: 11107B 14.3 minutes

 11 strokes 7. 9 minutes

Table 5

 As a result, the A-1544 / pIJDMG strain transformed with the plasmid containing psmA and psmB showed about 2.7 times the conversion activity in a 12-hour reaction as compared to the original A-1544 strain. This suggests that self-cloning of the psmA protein psmB can contribute to the conversion of the macrolide compound 11107B to 11107D.

 Example 7: Determination of the nucleotide sequence of a gene derived from Streptomyces' SP Mer-11107 strain (FE BP-7812)

 (1) Preparation of Streptomyces 'SP Mer-11107 strain chromosome 0'

 The medium consisting of glucose 1%, malt extract 0.4% and yeast extract 1% was inoculated with Mer-11107 strain and cultured at 28 ° C for 3 days. The obtained culture was centrifuged at 3000 rpm for 10 minutes to collect the cells. Chromosomal DNA was prepared from the cells using a Blood & Cell Culture kit (QIAGEN).

 (2) Cloning of partial sequence of DNA encoding protein having hydroxylation activity at position 16 of macrolide compound 11107

 Streptomyces cericolor 3 (2) cytochrome? A mix / primer (5Dm-3F (SEQ ID NO: 4) and 5D-1R (SEQ ID NO: 12)) was designed and created with reference to the amino / acid sequence estimated to be 450 ($ 10505). .

A mixed base S (= C + G), Y (= C + T) was used to increase the reactivity in consideration of codon fluctuation. - Next, a PCR reaction was performed using the two primers (5Dm-3F and 5D-1R) and the chromosomal DNA of the Mer-11107 strain obtained in (1) above as a template. The PCR reaction was performed using Takara LA Taq (Takara Shuzo) and a PCR amplifying device (Biometra T Gradient) at 98 ° C for 20 seconds for denaturation, 50 ° C for 2 minutes for annealing, and 68 ° C for 30 minutes for elongation. A three-step reaction of 35 seconds was repeated 35 seconds. As a result, a DNA fragment having a size of about 300 bp (hereinafter referred to as DNA fragment-A2) was amplified. This DNA fragment-A2 is likely to be a part of the DNA encoding the protein having hydroxylation activity. The DNA fragment-A2 amplified by the PCR reaction was recovered from the reaction solution by SUPREC PCR (Takara Shuzo).

 Next, in order to obtain a sufficient amount of the obtained DNA fragment -A2 to analyze the nucleotide sequence of A2, a DNA ligation kit ver. 2 (Takara Shuzo) was added to the plasmid vector pT7Blue T (Novagen). Was used to ligate the DNA fragment-A2 to transform Escherichia coli JM109 strain. Then, ampicillin (50 g / mL), X-gal (5-bromo-4-chloro-3- indoly β-D-galactoside; 40 μg / mL) IPTG (isopropyl-β-D-thiogalactopyranoside) Transformed L-Broth agar medium (1.0% batatotryptone, 0.5% yeast extract, 0.5% NaCl, 1.5% agar) containing 100 μL). Selected. The colonies of the transformed Escherichia coli thus isolated were cultured in an L-Broth liquid medium (1% noctotryptone, 0.5% yeast extract, 0.5% NaCl) containing ampicillin (50 μg / mL). Plasmid DNA was separated and purified from the grown cells of the transformed Escherichia coli using a plasmid purification kit (QIAfilter Plasmid Midi Kit, QIAGEN) to obtain a certain amount of DNA fragment-A2.

 (3) Analysis of nucleotide sequence of cloned DNA fragment-A2

 The nucleotide sequence of the DNA fragment-A2 obtained in (2) above was analyzed using a DNA nucleotide sequence analyzer (PE

Biosystems 377XL) was used to analyze the dye terminator using the 'cycle' sequence method. As a result of the nucleotide sequence analysis, the DNA fragment-A2 amplified by the PCR reaction was measured to be about 300 bp by electrophoresis, but the nucleotide sequence analysis revealed that it was exactly 325 bp (SEQ ID NO: Bases 237 to 1161). At both ends of the cloned 325 bp DNA sequence, DNA sequences corresponding to the two kinds of primers used in the PCK reaction were found, so that the DNA fragment -A2 was Kind of It was revealed that the DNA was specifically amplified by the primers (5Dm-3F and 5D-1R).

 (4) Analysis of the region around DNA fragment -A2,

As described above, since the partial sequence of the DNA encoding the protein having hydroxylation activity derived from the Mer-11107 strain was determined, it was determined by inverse PCR (Cell Engineering, Vol. 14, p. 591-593, 1995). The base sequence of the peripheral region extending upstream and downstream of the cloned fragment was extensively cloned and sequenced. That is, the chromosome DNA of the liter-11107 strain (see (1)) was digested with a restriction enzyme BamHI in a K buffer (50 mM Tris-HC1, pH 8.5, 10 mM MgCl ₂ , ImM dithioslate, 100 mM KC1) using H buffer. Each solution was digested with a restriction enzyme Sail in a solution (50 mM Tris-HC1, pH 7.5, 10 mM MgCl ₂ , ImM dithiothreitol, 100 mM NaCl). Each of the obtained restriction enzyme-cleaved DNA fragments was self-cyclized using DNA Ligation Kit ver. 2 (Takara Shuzo).

 On the other hand, primers (7PIN-2F (SEQ ID NO: 13) and 6PIN-2R (SEQ ID NO: 7)) were designed and prepared from the base sequence of DNA fragment-A2.

 Next, a PCR reaction was performed using these two primers (7PIN-2F and 6PIN-2R) and the chromosomal DNA of the self-circularized Mer-11107 strain as a template. The PCR reaction was performed using Takara LA Taq (Takara Shuzo) and a PCR amplifier (Biometra T Gradient), denaturation at 98 ° C for 20 seconds, annealing and extension at 68 ° C for 5 minutes 35 two-step reactions 35 times Repeated.

 As a result, a DNA fragment (DNA fragment-B2) with a size of about 1.3 kbp and a DNA fragment (DNA fragment-C2) with a size of about 1.4 kbp were amplified. There is a possibility that the DNA is a DNA encoding a DNA and a DNA having a DNA sequence including upstream and downstream regions thereof.

DNA fragment-B2 and DNA fragment-C2 were recovered from the PCR amplification reaction solution by SUPREC PCR (Takara Shuzo). Next, for the obtained DNA fragment-B2 and DNA fragment-C2, a plasmid vector pT7Blue T (Novagen Inc.), DNA Ligation kit ver. 2 (Takara Shuzo), Escherichia coli, 109 strains, and a plasmid purification kit (QIAfilter Plasmid Midi Kit, QIAGEN). As a result, a certain amount of each DNA fragment was obtained. .

 (5) Nucleotide sequence analysis of DNA fragment-B2 (about 1.3 kbp size) and DNA fragment-C2 (about 1.4 kbp size)

 The nucleotide sequences of DNA fragment-B2 and DNA fragment-C2 obtained in (4) above were solved by a DNA terminator one cycle cycle sequence method using a DNA sequence analyzer (PE Biosystems 377XL). The nucleotide sequence was analyzed in this manner, and information on the nucleotide sequence of 2329 bp shown in SEQ ID NO: 2 was obtained from the DNA fragment-B2 and DNA fragment-C2 sequences.

 A search for the open 'reading' frame (0RF) in the 2329 bp revealed that two proteins were encoded. A BLAST search of the amino acid sequences of these proteins revealed that 0RF (hereinafter referred to as bp mAb) encodes a protein consisting of 395 amino acids with high homology to cytochrome P450 from base 420 to base 1604 of SEQ ID NO: 2. There was). BpmA has the highest homology to the amino acid sequence of psmA isolated from the A-1544 strain (67.4% homology), and relatively high homology to Streptomyces griseus cytochrome P450soy (SoyC). (Homology 64.8%). This suggests that bpmA is likely to encode cytochrome P45Q-type hydroxylase.

 Immediately downstream of bpmA (base 1643 to base 1834 of SEQ ID NO: 2), 0RF (hereinafter referred to as bpmB) encoding a protein having high homology to 3Fe-4S type ferredoxin was present. The protein encoded by bpmB consists of 64 amino acids and has the highest homology (81.0% homology) with the amino acid sequence of psmB isolated from A-1544 strain. 3 (2) cytochrome? It also had relatively high homology (76.2%) to the amino acid sequence presumed to be ferredoxin immediately downstream of the amino acid sequence presumed to be 450 (? 10505). Therefore, it was thought that bpmB was responsible for electron transfer and hydroxylated with bpmA.

 Example 8: Preparation of transformant having bpmA and bpmB

(1) Preparation of DNA fragment containing both bpmA and bpmB derived from Mer-11107 strain Referring to the nucleotide sequence of SEQ ID NO: 2 analyzed in Example 7, a primer 07- NdeF (SEQ ID NO: 14) and 5, Spel at the end The site-added primer 07-SpeR (SEQ ID NO: 15) was designed and created. Next, a PCR reaction was carried out using these two primers (07-NdeF and 07-SpeR) and the chromosomal DNA of the Mer-11107 strain obtained in Example 7 (1) as a template. The PCR reaction was performed using Takara LA Taq (Takara Shuzo) and a PCR amplifying device (Bioraetra T Gradient). Denaturation was performed at 98 ° C for 20 seconds and annealing and extension were performed at 68 ° C for 2 minutes. Repeated times. As a result, a DNA fragment of about 1.5 kbp in size containing bpmA and bpmB (hereinafter referred to as DNA fragment-D2) was amplified. DNA fragment-D2 was recovered from this PCR amplification reaction solution by SUPREC PCR (Takara Shuzo).

 (2) Construction of plasmid pTC-D07

 pT7NS-CamAB (see W003 / 087381) in H buffer (50 mM Tris-HC1, pH 7.5, 10 mM)

The plasmid was digested with restriction enzymes Ndel and Spel in MgCl ₂ , ImM dithiothreitol, lOOmM NaCl) to obtain a plasmid digest. Similarly, the DNA fragment-D2 obtained in the previous section (1) is digested with restriction enzymes Ndel and Spel, and the obtained DNA fragment-D2 digest and plasmid digest are digested with DNA Ligation Kit ver. 2 (Takara Shuzo). And ligated. As a result, a plasmid (referred to as plasmid pTC-D07) having a size of about 9.5 kbp in which the DNA fragment -D2 containing both bpmA and bpmB and the plasmid pT7NS-CamAB were ligated was constructed. .

 (3) Preparation of E. coli transformant BL21 (DE3) / pTC-D07

 Escherichia coli BL21 (DE3) competent cells (Novagen) were transformed using the plasmid pTC-D07 prepared in (2) above. Thus, Escherichia coli BL21 (DE3) / pTC-D07 strain transformed with plasmid pTC-D07 was obtained.

 Example 9: Conversion of macrolide-based compound 11107B to 11107D by Escherichia coli transformant having bpmA and bpmB

 The transformed E. coli BL21 (DE3) / pTC-D07 strain obtained in Example 8 (3) and

BL21 (DE3) / pT7NS-CamAB strain frozen seeds containing 50 g / mL ampicillin in L-Broth medium ( ^1.0 /. Batatotryptone, 0.5% yeast extract, 0.5% NaCl) 3 mL Was inoculated into a 15-mL test tube containing, and shake-cultured at 37 ° C for 20 hours. Add 500 L of this seed culture to a 250 mL volume containing 50 mL of L-Broth medium (1.0% batatotryptone, 0.5% yeast extract, 0.5% NaCl) containing 50 μg / mL ampicillin. Inoculate Erlenmeyer flask and shake culture at 32 ° C for 4 hours After that, lOOmM IPTG (isopropyl- -D-thiogalactopyranoside) ¾f 50 µ 80 mg / mL 5-amino levulinic acid was added in order of 50 IL, and cultured with shaking at 32 ° C for 5 hours. The resulting culture was centrifuged (5000 rpm, 10 minutes) to collect the cells. This was suspended in 1.75 mL of lOOmM phosphate buffer (pH 6.1), and 250 µL of 80% glycerol and 12.5 µL of 40 mg / mL macrolide compound 11107B were added thereto. The conversion reaction solution thus obtained was reacted at 28 ° C. for 24 hours. 400 of the reaction solution was extracted with 600 ^ L of methanol, and the amounts of macrolide compounds 11107B and 11107D were measured by HPLC. Table 6 shows the measurement results. The detailed conditions of HPLC are shown below.

 Analyzer: Shimadzu HPLC ΙΟΑνρ

 Column: Develosil 0DS UG-3 (φ4.6 marauder X 250mm 3 β η)

 Mobile phase: 45% to 55% methanol (0 to 5 minutes)

 55% methanol (5-13 minutes)

 55% to 70% methanol (13 to 17 minutes)

 70% methanol (17-21 minutes)

 45% methanol (21-25 minutes)

 Flow rate: 1.2 mL / min

 Detection: UV240nm

 Injection capacity: 5 L

 Column temperature: 40 ° C

 Analysis time: 25 minutes

 Retention time: 11107B 12.2 minutes

 . 11107D 4.2 minutes

Table 6

As a result, the control of E. coli BL21 (DE3) / pT7NS-CamAB strain Whereas the peak of the I de compound 11107D was obtained, in bpmA and including BL21 a bpmB (DE3) / pTC-D07 strain, the peak of the macrolide compound 1110 ⁷ D was obtained. This suggests that bpmA and bpmB are involved in the conversion of macrolide compound 11107B to 11107D.

 Example 10: Determination of base sequence of gene derived from A-1560 strain (FERM BP-10102)

 (1) Preparation of DNA of chromosome A-1560

 A-1560 strain was inoculated into a medium consisting of glucose 1%, malt extract 0.4%, and yeast extract 1%, and cultured at 28 ° C for 3 days. The obtained culture was centrifuged at 3000 rpm for 10 minutes to collect the cells. Chromosomal DNA was prepared from the cells using a Blood & Cell Culture kit (QIAGEN).

 (2) Cloning of partial sequence of DNA encoding protein having hydroxylation activity at position 16 of macrolide compound 11107

 Streptomyces cericolor 3 (2) cytochrome? Mix 'primers (5Dm-3F (SEQ ID NO: 4) and 5Dm-2R (SEQ ID NO: 16)) were designed and prepared with reference to the amino acid sequence estimated to be 450 (10-50).

 A mixed base S (= C + G), Y (= C + T) was used to increase the reactivity in consideration of codon fluctuation.

 Next, a PCR reaction was performed using these two primers (5Dm-3F and 5Dm-2R) and the chromosomal DNA of the A-1560 strain obtained in the above (1) as a template. The PCR reaction was performed using Takara LA Taq (Takara Shuzo) and a PCR amplifier (Biometra T Gradient) for denaturation at 98 ° C for 20 seconds, annealing at 50 ° C for 2 minutes, and elongation at 68 ° C for 30 seconds. The three-step reaction was repeated 35 times, and as a result, a DNA fragment of about 750 bp in size (hereinafter, DNA fragment-A3) was amplified. This DNA fragment-A3 is likely to be part of the DNA encoding a protein having hydroxylic activity. The DNA fragment-A3 amplified by the PCR reaction was recovered from the reaction solution by SUPREC PCR (Takara Shuzo).

Next, in order to obtain a sufficient amount of DNA fragment-A3 to analyze the base sequence of the obtained DNA fragment-A3, DNA ligation kit ver. 2 (Takara Shuzo) was added to plasmid vector pT7Blue T (Novagen). Ligated DNA fragment-A3 using E. coli and transformed E. coli JM109 strain (Stratagene) Changed. Then, ampicillin (50 μg / mL), X-gal (5-bromo-4-chloro-3-indolyl-j3-D-galactoside; 40 μg / mL), IPTG (isopropyl-β-D-thiogalactopyranoside; Select transformed Escherichia coli using L-Broth agar medium (100% / iM) (1.0% batatotributone, 0.5% yeast extract, 0.5% NaCl, 1.5% agar) did. The colonies of the transformed Escherichia coli thus isolated were cultured in an L-Broth liquid medium (1% bactotryptone, 0.5% yeast extract, 0.5% NaCl) containing ampicillin (50 μg / mL). Plasmid DNA was separated and purified from the grown cells of the transformed Escherichia coli using a plasmid purification kit (QIAfilter Plasmid Midi Kit, QIAGEN) to obtain a certain amount of DNA fragment-A3.

 (3) Analysis of nucleotide sequence of DNA fragment-A3

 The DNA sequence of the DNA fragment-A3 obtained in (2) above was analyzed using a DNA sequence analyzer (PE

Using Biosystems 377XL), the analysis was carried out by the dye terminator-cycle method. As a result of the nucleotide sequence analysis, the DNA fragment-A3 amplified by the PCR reaction was measured to be approximately 750 bp by electrophoresis. However, the nucleotide sequence analysis revealed that the DNA fragment-A3 was correctly 741 bp (SEQ ID NO: 3, bases 616 to 1356). Since DNA sequences corresponding to the two types of primers used in the above PCR reaction were found at both ends of the cloned 741 bp DNA sequence, the DNA fragment-A3 was It was revealed that the primers were specifically amplified by the different types of primers (5Dra-3F and 5Dm-2R).

 (4) Analysis of DNA fragment-A3 peripheral region

As described above, since the partial sequence of the DNA encoding the protein having hydroxylation activity derived from the A-1560 strain was determined, it was cloned by inverse PCR (Cell Engineering, Vol. 14, p. 591-593, 1995). We amplified, cloned, and sequenced the base sequence in the peripheral region extending upstream and downstream of the fragment. That is, the chromosomal DNA of the A-1560 strain (see (1)) was subjected to L-buffering with the restriction enzyme BaraHI in K buffer (50 mM Tris-HCl, pH 8.5, 10 mM MgCl ₂ , ImM dithiothreitol, lOOmM KC1). liquid with _{(10mM Tris- HCl, P H7.} 5, 10mM MgCl 2, ImM dithiothreitol I torr) restriction enzyme Kpnl during, Eta buffer solution (50raM Tris-HCl, pH7. 5, 10mM MgCl 2, ImM Jichiosurei Torr, lOOtnM NaCl) Each was digested with the restriction enzyme Sail. Each of the obtained restriction enzyme-cleaved DNA fragments was self-cyclized using DNA Ligation Kit ver. 2 (Takara Shuzo).

 On the other hand, primers (5PIN-2F (SEQ ID NO: 17) and 6PIN-2R (SEQ ID NO: 7)) were designed and prepared from the nucleotide sequence of DNA fragment-A3.

 Next, PCR was carried out using these two primers (5HN-2F and 6PIN-2R) and the chromosomal DNA of the self-cyclized A-1560 strain as a template. The PCR reaction was performed using Takara LA Taq (Takara Shuzo) and a PCR amplifier (Biometra T Gradient), denaturing at 98 ° C for 20 seconds, annealing and elongation at 68 ° C for 5 minutes, two-step reaction 35 times Repeated.

 As a result, a DNA fragment of about 4.5 kbp (DNA fragment-B3) and a DNA fragment of about 3. Okbp (DNA fragment-C3 and a DNA fragment of about 1.7 kbp (DNA fragment- D3) was amplified, but these are likely to be DNA encoding a protein having hydroxylation activity and DNA having a DNA sequence containing upstream and downstream regions thereof.

 DNA fragment-B3 and DNA fragment-C3 and DNA fragment-D3 were

Collected by SUPREC PCR (Takara Shuzo). Next, for the obtained DNA fragment-B3, DNA fragment-C3 and DNA fragment-D3, plasmid vector pT7Blue was obtained in the same manner as in the above (2), in order to obtain sufficient DNA fragments for nucleotide sequence analysis. Using T (Novagen), DNA Ligation kit ver. 2 (Takara Shuzo), E. coli JM109 strain, and a plasmid purification kit (QIAfilter Plasmid Midi Kit, QIAGEN), a certain amount of each DNA fragment was obtained.

(5) DNA fragment-B3 (about 4.-bp size), DNA fragment-C3 (about ^3. Okbp size) and DNA fragment-D3 (about 1.7 kbp size) nucleotide sequence analysis

Analyze the nucleotide sequence of DNA fragment-B3, DNA fragment-C3 and DNA fragment-D3 obtained in the previous section ( ₄ ) using a DNA terminator-cycle-sequence method using a DNA sequence analyzer (PE Biosystems 377XL). did. The nucleotide sequence was analyzed in this way, and information on the 1860 bp base sequence shown in SEQ ID NO: 3 was obtained from the sequences of DNA fragment-B3, DNA fragment-C3 and DNA fragment-D3.

A search for the open 'reading' frame (0RF) in the 1860 bp revealed that two proteins were encoded. The key to these proteins PT / JP2004 / 017906

The amino acid sequence result of the search by BLAST search, 404 pieces encoding a protein comprising the amino acid to ¾0RF that has high homology with cytochrome P450 basic 172～ nucleotide 1383 of SEQ ID NO: 3 (hereinafter referred to t _P) is present did. And is tpmA a cytochrome of Streptomyces sericolor 3 (2)? 4500 ^? 10505) with the highest homology (77.4% homology) and also with the amino acid sequence of psmA isolated from A-1544 strain ( Nematode same sex 76.6%). This suggests that tpmA is likely to be a gene encoding cytochrome P450-type hydroxylase.

Immediately downstream of tpmA (base 1399 to base 1593 of SEQ ID NO: 3), 0RF (hereinafter referred to as tpmB) ′ encoding a protein having high homology to 3Fe-4S type ferredoxin was present. The protein encoded by tpmB consists of 65 amino acids, has the highest homology to the amino acid sequence of psmB isolated from strain A-1544 (81.0% homology), and has Streptomyces' Sericolor 3 (2 ) cytochrome 1 ⁵ 4500 ^? 10505) and had a high homology to Amino acid sequence deduced immediately downstream of ferredoxin Amino acid sequence deduced (82.5%). Therefore, tpmB was thought to encode ferredoxin, which is responsible for electron transfer and hydroxylates with tpmA.

 Example 11 1: Preparation of Transformant Having tpmA and tpmB

 (1) Preparation of DNA fragment containing both tpmA and tpmB from A-1560 strain

 Referring to the nucleotide sequence of SEQ ID NO: 3 analyzed in Example 10, the 5 '

A primer tpm-NdeF (SEQ ID NO: 18) to which an Ndel site was added and a primer tpm-SpeR (SEQ ID NO: 19) to which an Spel site was added at the 5 'end were designed and prepared. Next, a PCR reaction was performed using these two primers (tpm-NdeF and tpm-SpeR) and the chromosomal DNA of the A-1560 strain obtained in Example 10 (1) as a template. The PCR reaction was performed using Takara LA Taq (Takara Shuzo) and a PCR amplification device (Biometra T Gradient). Denaturation was performed at 98 ° C for 20 seconds and annealing and extension were performed at 68 ° C for 2 minutes. I turned around again.

 As a result, a DNA fragment of about 1.5 kbp including tpmA and tpmB (hereinafter, referred to as DNA fragment-E3) was amplified. DNA fragment-E3 was recovered from this PCR amplification reaction solution by SUPREC PCR (Takara Shuzo).

(2) Construction of plasmid pTC-tpmAB 7906 pT7NS-CamAB (see W003 / 087381) in H buffer (50 mM Tris-HC1, pH 7.5, 10 mM

It was digested with the restriction enzymes Ndel and Spel in MgCl ₂₎ ImM dithiothreitol, lOOmM NaCl) to obtain a plasmid digest. Similarly, the DNA fragment-E3 obtained in (1) above is digested with restriction enzymes Ndel and Spel, and the obtained DNA fragment-E3 digest and plasmid digest are combined with DNA Ligation Kit ver. 2 (Takara Shuzo) Were connected. As a result, a plasmid of about 9.5 kbp (referred to as plasmid pTC-tpmAB) in which the DNA fragment containing both tpmA and tpmB-E3 and the plasmid pT7NS-CamAB were linked was constructed. Was done.

 (3) Preparation of E. coli transformant BL21 (DE3) / pTC-tpmAB

 E. coli BL21 (DE3) competent cells (Novagen) were transformed using the plasmid pTC-tpmAB prepared in Example 11 (2). Thus, Escherichia coli BL21 (DE3) / pTC-tpmAB strain transformed with plasmid pTC-tpmAB was obtained.

 Example 12 Conversion of 11107B to 11107D by E. coli Transformants with tpmA and tpmB

 The transformed E. coli BL21 (DE3) / pTC-tpmAB strain obtained in (3) above, and

BL21 (DE3) / pT7NS-CamAB frozen seeds were grown on 3 mL of L-Broth medium (1.0% bactotryptone, 0.5% yeast extract, 0.5% NaCl) containing 50 μg / mL ampicillin. The cells were inoculated into a 15 mL test tube and shake-cultured at 37 ° C for 20 hours. Add 500 L of this seed culture to a 250 mL volume of 50 mL of L-Broth medium (1.0% bactotryptone, 0.5% yeast extract, 0.5% NaCl) containing 50 μg / raL of ampicillin. After inoculating the flask in an Erlenmeyer flask and shaking the culture at 32 ° C for 4 hours, add lOOmM IPTG (isopropyl-β-D-thiogalactopyranoside) ¾r 50 μ 80 mg / mL And shaked for 5 hours. The resulting culture was centrifuged (5000 rpm, 10 minutes) to collect the cells. This was suspended in 1.75 mL of 100 mM phosphate buffer (pH 6.1), and 250 μL of 80% glycerol and 12.5 L of 40 mg / mL 11107B were added thereto. The conversion reaction solution thus obtained was reacted at 28 ° C. for 24 hours. 400 L of the reaction solution was extracted with 600 μL of methanol, and the amounts of 11107B and 11107D were measured by HPLC. Table 7 shows the measurement results. The detailed conditions of HPLC are shown below.

 Analyzer: Shimadzu HPLC ΙΟΑνρ

Column: Develosil 0DS UG-3 (4.6mmx250mm 3 zm) Mobile phase: 45% to 55% methanol (0 to 5 minutes)

 55% methanol (5-13 minutes)

 55% -70% methanol (13-1a)

 70% methanol (17-21 minutes)

 45% methanol (21-25 minutes)

 Flow rate: 1.2 mL / min

 Detection: UV240nm

 Injection capacity 5 L

 Column temperature: 40 ° C

 Analysis time: 25 minutes

 Retention time: 11107B 12.2 minutes

 11107D 4.2 minutes

Table 7

As a result, BL21 in E. coli BL21 (DE3) / _P T7NS- CamAB strain as a control includes whereas not obtained peak of 11107D, a tpmA and tpmB (DE3) / pTC - In tpmAB strain, the peak of 11107D was gotten. This suggests that tpmA and tpmB are involved in the conversion of 11107B to 11107D.

Example 1 · 3: Conversion of 11107H into 11107CB represented by the following formula using a self-cloning strain

 11107CB

 (1) Preparation of transformant reaction solution

Stabilizer rose 2.0%, glucose ₂ · 0 ° / ο, soy flour (Honen Soipuro) 2.0% yeast extract 0.5%, to prepare a medium pH 7. 4 consisting of CaC0 ₃ 0. 32% Then, 25 mL of the medium was placed in a 250-raL triangular flask, and sterilized by heating at 121 ° C for 20 minutes. After adding thiostrepton to this medium to a final concentration of 25 mg / L, the A-1544 / pIJOMG strain was inoculated at 1% from the frozen seed mother, and the seed mother was cultured at 28 ° C and 220 rpm for 3 days. 1% of this seed culture was added to a medium having the same composition, and main culture was performed at 28 ° C and 220 rpm for 2 days. After completion of the main culture, the cells were collected from the culture by centrifugation, and suspended in 20 mL of a pH 6.5 phosphate buffer. Substrate 11107H (100 g / L DMS0 solution) was added to the cell suspension to a final concentration of 2000 mg / L, and a conversion reaction was performed at 28 ° C and 220 rpm for 16 hours.

 (2) Acquisition of Macmouth Ride Compound 11107CB from Transformant Reaction Solution

Cells were separated by centrifugation from the conversion reaction solution (six flasks) subjected to the same operation, and the centrifuged supernatant was extracted twice with an equal volume of ethyl acetate. After concentrating the extract, 4017906

Purification by chromatography (MERCK Silicagel 60 F254 '0.5 developing solution; toluene: acetone = 1': 1) gave 119.5 mg of 11107CB.

ESI-MS m / z 573 (M + Na) +

-丽R scan Bae spectrum _{(CD 3 0D, 500MHz):} δ ppm ( integration, multiplicity, coupling constant J (Hz)):

0.81 (3H, d, J = 6.7Hz), 0.89 (3H, d, J = 7.0), 0.94 (3H, t, J = 7.4Hz), 1.25 (3H, s), 1.30-1.20 (IH, m) , 1.33 (3H, s), 1.55-1.40 (2H, m), 1.65 (lH, dd, J = 6.3, 14.0Hz), 1.75 (3H, s), 1.88 (1H, dd, J = 5.4, 14.0Hz ), 2.07 (3H, s), 2.68-2.40 (4H, m),

2.89 (IH, m), 3.5l (lH, m), 4.51 (1H, m), 4.97 (lH, d, J = 8.6Hz),

 4.99 (IH, d, J = 9.3Hz), 5.30 (IH, dd, J = 9.7, 15.2Hz), 5.52 (IH, dd, J = 9.4, 15.2Hz),

5.58 (IH, dd, J = 1.9, 15.5Hz), 5.78 (IH, dd, J = 2.8, 15.5Hz), 5.85 (IH, d, J = 15.3Hz),

6.07 (IH, d, J = ll.0Hz), 6.51 (IH, dd, J = ll.0, 15.3Hz)

 Example 14: Using a self-cloning strain, 11107L represented by the following formula:

Conversion to 11107CG

 11107CG

(1) Preparation of transformant reaction solution Prepare a pH 7.4 culture medium consisting of 2.0% Stabilose, 2.0% glucose ^ο / _ο , 2.0% soybean flour (Honen Soypro), 0.5% yeast extract, and 0.32% CaC03, and add 25mL of medium to a 250mL triangular flask. And heat sterilized at 121 ° C for 20 minutes. After adding thiostrepton to this medium to a final concentration of 25 _mg / L, the A-1544 / pIJDMG strain was inoculated at 1% from the frozen seed, and the seed was cultured at 28 ° C, 220 rpm for 3 days. . 1% of this seed culture was added to a medium having the same composition, and main culture was performed at 28 ° C and 220 rpm for 2 days. After completion of the culture, the cells were collected from the culture broth by centrifugation, and suspended in 20 mL of a pH 6.5 phosphate buffer. To this cell suspension was added 11107 L of a substrate (100 g / L DMS0 solution) so as to have a final concentration of 1600 _mg / L, and a conversion reaction was performed at 28 ° C. and 220 rpm for 16 hours.

(2) Acquisition of Macmouth Ride Compound 11107CG from Transformant Reaction Solution

 Cells were separated from this conversion reaction solution by centrifugation, and the centrifuged supernatant was extracted twice with an equal volume of ethyl acetate. After concentrating the extract, thin-layer chromatography (MERCK

Purification was performed using Silicagel 60 F254 '0.25 mm developing solution; toluene: acetone = 1: 1) to obtain 25 mg of 11107CG.

ESI-MS m / z 633 (M + Na) +

-顧R spectrum _{(CD 3 0D, 500MHz):} δ ppm ( integration, multiplicity, coupling constant J (Hz)): 0.88 ( 3H, d, J = 6.7Hz), 0.90 (3H, d, J = 7.0Hz), 0.94 (3H, d, J = 7.4Hz), 1.18 (3H, s), 1.30-1.20 (1H, m), 1.34, (3H, s), 1.56-1.40 (2H, m), 1.66 (1H, dd, J = 6.2, 14.0Hz), 1.79-169 (2H, m), 1.81 (3H, d, J = 1.0Hz), 1.86 (1H, dd, J = 5.4, 14.0Hz),

2.05 (3H, s), 2.08 (3H, s), 2.52 (1H, dd, J = 4.2,15.2Hz), 2.64-2.55 (1H, m), 2.67 (1H, dd, J = 2.2, 7.9Hz) '2.78 (1H, dd, J = 3.0, 15.2Hz),

2.90 (1H, dt, J = 2.2, 5.6Hz), 3.52 (1H, dt, J = 4.4, 8.8Hz), 3.75 (1H, m),

 4.98 (1H, dd, J = 2.8, 11.3Hz), 5.08 (1H, d, J = 9.7Hz), 5.13 (1H, d, J = 9.6Hz),

 5.61 (1H, dd, J = 9.9, 15.2Hz), 5.75 (1H, dd, J = 9.7, 15.2Hz), 5.88 (1H, d, J = 15.3Hz), 6.13 (1H, d, J = ll. 0Hz), 6.54 (1H, dd, J = 11.0, 15.3Hz) Industrial applicability

Use of a transformant transformed with a plasmid carrying the DNA of the present invention As a result, it is possible to efficiently produce a 12-membered ring macrolide compound having excellent antitumor activity and excellent stability in an aqueous solution and having a hydroxyl group at the 16-position.

Claims

Claims formula. (I)

 11107B The macrolide compound represented by (i) (hereinafter referred to as macrolide compound 11107B)

Formula (II)

 11107D DN involved in biological conversion to 16-hydroxylated macrolide compound represented by (π)

Α, an isolated pure DNA comprising a DNA partially or wholly encoding a protein having 16-hydroxylase activity or ferredoxin or a variant thereof.

 2. The DNA according to claim 1, which is represented by the following (a;), (b) or (c).

(a) DNA encoding a protein having a hydroxylase activity at position 16 of macrolide compound 11107B, comprising a continuous base sequence from base 1322 to base 2548 of SEQ ID NO: 1, base 420 of SEQ ID NO: 2 Contiguous base sequence from to base 1604 And a DNA selected from the group consisting of a continuous base sequence from base 172 to base 1383 of SEQ ID NO: 3.

 (b) a variant of the DNA shown in the above (a),

 (i) hybridizing with the DNA shown in (a) under stringent conditions, and

 (ii) DNA encoding a protein having a 16-position hydroxylase activity of macrolide compound 11107B.

 (c) does not hybridize under stringent conditions with the DNA shown in (a) due to degeneracy of gene codons, but does not hybridize to a protein encoded by the DNA shown in (a) or (b) DNA encoding a protein having the same amino acid sequence as

 3. A protein encoded by the DNA according to claim 2.

 4. An autonomously replicating or integrating replicative recombinant plasmid carrying the DNA of claim 2.

 5. A transformant transformed with the recombinant plasmid according to claim 4.

 6. Encoding a protein having a 16-position hydroxylase activity of macrolide compound 11107B, wherein the DNA of claim 2 or a part thereof is used as a probe or primer. Methods for isolating DNA.

 7. The DNA according to claim 1, which is represented by the following (d), (e) or (f).

 (d) DNA encoding ferredoxin, a continuous base sequence from base 2564 to base 2761 of SEQ ID NO: 1, a base sequence from base 1643 to base 1834 of SEQ ID NO: 2, and SEQ ID NO: DNA selected from the group consisting of a continuous base sequence from base 3399 to base 1593 of 3;

 (e) a modified form of the DNA represented by (

 (i) hybridizing with the DNA shown in (d) under stringent conditions, and

 (ii) DNA encoding a protein having a ferredoxin function.

(f) Due to the degeneracy of gene codons, the DNA and stringent shown in (d) above DNA that does not hybridize under mild conditions but has the same amino acid sequence as the protein encoded by the DNA represented by (d) or (e). .

 8. A protein encoded by the DNA according to claim 7.

 9. An autonomously replicating or integrated replicating recombinant plasmid carrying the DNA of claim 7.

 10. A transformant transformed with the recombinant plasmid according to claim 9.

 1 1. A method for isolating a DNA encoding a protein having a ferredoxin function, comprising using the DNA of claim 7 or a DNA thereof as a probe or a primer.

 12. The transformant according to claim 5 or claim 10 is cultured in a medium, and the transformant grown during or after the culturing is expressed by the formula (II)

(In the formula,

R ¹² , R ^16b , R ^17a , R ^17b , R ¹⁸ , R ^20a , R ^20b , R ^21a and R ^{2 are the} same or different,

 (1) hydrogen atom,

 (2) C—alkyl group which may have a substituent,

 (3) One OR (where R is

1) hydrogen atom, Which may have a substituent,

 2) — alkyl group,

3) C ₇ one ₂₂ Ararukiru group,

 4) 5- to 14-membered heteroaryloxyalkyl group,

5) C ₂ one ₂₂ Arukanoiru group,

6). ₇ _ ₁₅ aroyl group,

7) C ₃ — ₂₃ unsaturated alkanoyl group,

8) _C〇R ^C. (In the formula, Rc. May have a substituent,

 8-1) 5- to 14-membered heteroaryl group,

'8- 2) — ₂₂ alkoxy group,

8-3) unsaturated C ₂ - ₂₂ alkoxy group,

8-4) C ₆ — ₄ aryloxy group,

 8-5) 5-membered ring to 14-membered heteroaryloxy group,

 Or

 8- 6) 3- or 14-membered nitrogen-containing non-aromatic heterocyclic ring),

9) phenol ₂₂ sulfonyl group,

10) C ₆ — ₁₄ arylsulfonyl group

 Or

1 1) One S i R ^sl R ^{s 2} R ^{s 3} (wherein, R ^sl , R ^{s 2} and R ^s3 are the same and represent a Jaralkyl group or a Faryl group),

(4) Halogen atom

Or

(5) One R ^M — NR ^N1 R ^N2

{Wherein, R ^M represents a single bond or —O—CO—;

R ^N1 and R ^N2 are

 1) same or different,

 1-1) hydrogen atom or

1-2) may have a substituent, (i) an alkyl group,

(ii) unsaturated C ₂ - ₂₂ alkyl group,

(iii) C ₂ one ₂₂ Arukanoiru group

(iv) C ₇ - ₁₅ Aroiru group,

(v) unsaturated C ₃ _ ₂₃ Arukanoiru group,

(vi) 0 ₆ _ ₁₄ reel group,

 (vii) a 5- to 14-membered heteroaryl group,

(viii) C ₇ - ₂₂ Ararukiru group,

(ix) one ₂₂ alkylsulfonyl group or

'(X) 〇 ₆ _ ₁₄ represents an arylsulfonyl group,

 Or

2) R ^N1 and R ^N2 are taken together with the nitrogen atom to form a 3- or 14-membered nitrogen-containing non-aromatic heterocyclic ring which may have a substituent;

R ^21a and R ^21b are joined together to form (i) a ketone structure (-0) or (ii) an oxime structure {= NOR. ^x (wherein, R. ^x may have a substituent, ₂₂ alkyl group, unsaturated C ₂ - ₂₂ alkyl groups, C ₆ - ₁₄ Ariru group, 5-membered ring to Teroarinore group to 14-membered ring or C ₇ - ₂₂ Ararukiru represents a group)} may be formed;

R ^16a represents ^a hydrogen atom;

R ^{21 c}

 (1) hydrogen atom or

 (2)

(Wherein, R ^22a , R ^22b and R ^22c are the same or different,

1) hydrogen atom, 2) C-6 alkyl group,

 3) -OR, wherein R has the meaning described above,

4) — R ^M — NR ^N1 R ^N2 (where R ^M , R ^N1 and R ^N2 have the above meanings) or

 5) Halogen atom

 Represents;

 Or

R ^{21 a} and either of R ^21b and R ^22a and partially turned Either Toga cord of R ^22b structure

May be formed;

G ^m

 (1) a group represented by the formula (GM-I)

{Where,

R ² and R ^{1 (3} are the same or different, to display the hydrogen atom _or an alkyl group;

R ^3a , R ^3b , R ^5a , R ^5b , R ^6a and R ^6b are the same or different,

1) hydrogen atom, 2) hydroxy group,

 3) may have a substituent,

3-1) ₂₂ alkyl groups,

3-2) — ₂₂ alkoxy groups,

3-3) C 64 ₄ aryloxy group

 3-4) 5- or 14-membered heteroaryloxy group,

3-5) C ₂ _ ₂₂ alkanoyloxy group,

3-6) 〇 ₇ _ ₁₅ aryloxy group

3-7) C ₃ - ₂₃ unsaturated alk Noi Ruo alkoxy group,

• 3-8) _OCOR ^e ° (where R ^c ° has the above meaning),

3-9) Ci- ₂₂ alkylsulfonyloxy group,

3-10) C ₆ — ₁₄ arylsulfonyloxy group

 Or

3-11) -OS i R ^sl R ^{s 2} R ^{s 3} (where R ^s R ⁵² and R ^{s 3} have the same meanings as above),

 4) Halogen atom

 Or

5) represents -R ^M -NR ^N1 R ^N2 , wherein R ^M , R ^N1 and R ^N2 have the above-mentioned meanings;

 Or

R ^5a and R ^5b may together form a ketone structure (= 0); or

R ^{6 a} and R ^{6 b} is MAY together form a connexion, spiro O carboxymethyl oxiranyl group or Ekisomechire emissions group; or,

R ^7a and R ^7b are the same or different and each represent a hydrogen atom or one OR ^H (wherein R ^H represents a hydrogen atom, a C ^ alkynole group or a C ₂ _ ₂₂ alkanoyl group)}, (2) Group represented by GM-II)

(Wherein R ² , R ^3a , R ^3b , R ^6a , R ^6b , R ^7a , R ^7b and R ¹⁰ are as defined in the formula (GM-1)),

 (3) a group represented by the formula (GM-III)

(Wherein R ² , R ^5a , R ^5b , R ^6a , R ^6b , R ^7a , R ^7b and R ¹⁰ are as defined in the formula (GM-I)),

 (4) Group represented by the formula (GM-IV)

(Wherein R ² , R ^6a , R ^7a , R ^7b and R ¹⁰ are as defined in the formula (GM-1). )

Or

 (5) group represented by the formula (GM-V)

(Wherein, R ² , R ^3a , R ^6a , R ^6b and R ¹ represent the same as defined in formula (GM-1))

With a macrolide compound represented by the formula (IV)

(Wherein, it represents ^{W, R 12, R 16b,} R 17a, R 17b, R 20a, R 20b, R 21a, and R ^21b, 11 ² Oyopi 0 ¹¹¹ defines the same meaning as formula (III))

A method for producing a 16-hydroxyl-hymic-mouth compound comprising converting into a 16-hydroxyl-hymachic-ride compound represented by the formula: .

 13. The production method according to claim 12, wherein the transformant is the transformant according to claim 5, wherein the transformant is a transformant having a DNA encoding fredoxin.

14. Expression (ΙΠ-a)

(Where

H. A H

5 = _-4 is double bond or single bond, W 'is double bond or

R ⁵ ′ represents a hydrogen atom or an acetyl group, R ⁶ ′ represents a hydrogen atom or a hydroxy group, and R ⁷ ′ represents a hydrogen atom or an acetyl group), a compound represented by the formula (IV-a)

C

(Where

^{. W ', R 5',} R 6 ' and R ^7' have the formula - method of producing according to claim 12, wherein the converting the definition is as defined above) compound of represented by the (III a) .

15. In converting a compound of the formula (III-a) into a compound of the formula (IV-a),

A compound wherein R ⁵ ′, R ⁶ ′ and R ⁷ ′ are hydrogen atoms,

 (2)

H, Q H

 5-4 is a single bond, W 'is

A compound in which R ⁵ ′ and R ⁶ ′ are a hydrogen atom, and R ⁷ ′ is an acetylino group,

 (3)

H. Eight

 5-4 is a single bond, W 'is

A compound wherein R ⁵ ′ and R ⁷ ′ are a hydrogen atom, and R ⁶ ′ is a hydroxy group,

 (Four)

5-4 is a single bond, W is

A compound in which R ⁵ ′ is a hydrogen atom, R ⁶ ′ is a hydroxy group, and R ⁷ ′ is an acetyl group, (5)

5-4 is a single bond,

A compound wherein W is a double bond and R ⁵ ′, R ⁶ ′ and R ⁷ ′ are hydrogen atoms,

 (6)

5-4 is a single bond,

A compound wherein W is a double bond, R ⁵ ′ and R ⁶ ′ are hydrogen atoms, and R ⁷ ′ is an acetyl group, (7).

5-4 is a single bond,

W is a double bond, R ⁵ ′ and R ⁷ ′ are hydrogen atoms, and R ⁶ ′ is a hydroxy group object,

 (8) is a single bond,

A compound in which W ′ is a double bond, R ⁵ ′ is a hydrogen atom, R ⁶ ′ is a hydroxy group, and R ⁷ ′ is an acetyl group,

 (9)

H ゝ H

 5 4 is a double bond, W, is

A compound wherein R ⁵ ′ and R ⁷ ′ are a hydrogen atom, and R ⁶ ′ is a hydroxy group,

 (Ten)

H ゝ H

 Is a double bond, w, is

A compound in which R ⁵ ′ is a hydrogen atom, R ⁶ ′ is a hydroxy group, and R ⁷ ′ is an acetyl group, (11)

H, eight H

 5-4 is a single bond, W 'is> ^

Compounds in which R ⁵ ′ is an acetyl group, R ⁶ ′ is a hydroxy group, R ⁷ ′ is a hydrogen atom, and

 (12)

H, eight H

 Is a single bond, w 'is ~ ^ <^

15. The production method according to claim 14, which is directed to a compound selected from the group consisting of a compound in which R ⁵ ′ is an acetyl group, R ⁶ ′ is a hydroxy group, and R ⁷ ′ is an acetyl group.

16. Use of the transformant according to claim ⁵ or 10 for production of a 16-hydroxylated macrolide compound.